An Albumin-Based Therapeutic Nanosystem for Photosensitizer/Protein Co-Delivery to Realize Synergistic Cancer Therapy.

Oxygen-dependent photodynamic therapy (PDT) is hindered by the limited availability of endogenous oxygen in solid tumors and low tumor accumulation of photosensitizers. Herein, we developed a biocompatible cancer-targeted therapeutic nanosystem based on cRGD conjugated bovine serum albumin (CBSA) co-loaded with a photosensitizer (chlorin e6, Ce6) and a therapeutic protein (cytochrome c, Cytc) for synergistic photodynamic and protein therapy. The nanosystem (Ce6/Cytc@CBSA) can target αVß3 integrin overexpressed cancer cells to improve tumor accumulation due to incorporation of cRGD. In the intracellular environment, Ce6 is released to produce toxic singlet oxygen upon near-infrared irradiation. At the same time, the therapeutic protein, Cytc, can induce programmed cell death by activating the downstream caspase pathway. Most importantly, Cytc with the catalase-like activity accelerates O2 generation by decomposing excess H2O2 in cancer cells, thereby relieving the PDT-induced hypoxia to enhance therapeutic efficacy. Both in vitro and in vivo studies reveal the significantly improved antitumor effects of the combined photodynamic/protein therapy, indicating that Ce6/Cytc@CBSA shows great potential in synergetic cancer treatments.

Cytochrome c injection induced embryo loss.

Cytochrome c has been used as first-aid in the clinic for organs which are lacking oxygen. But recent report show cytochrome c injection destroys dendritic cells (DCs) which play a pivotal role in feto-maternal tolerance. However, it is not clear whether cytochrome c injection causes abortion. The cytochrome c was injected by tail vein of mice at the Day 5.5 of pregnancy (E5.5) after mating with male BALB/c mice. The total number of implantations and resorption sites was recorded at the E12.5 in pregnant mice. Expression of interferon-γ, tumor necrosis-α interleukin (IL)-4, IL-10, IL-12 and transforming growth factor-ß in the mouse endometrium was measured by ELISA. Injection of cytochrome c via tail vein at the E5.5 induced fetal resorption at E12.5, and evoked an immune imbalance at the maternal-fetal interface. Notably, injection of mouse bone marrow-derived DCs (BM-DCs) rescued the cytochrome c-evoked embryo resorption. The present study suggests cytochrome c injection causes embryo resorption in mice, hinting caution regarding the use of cytochrome c in pregnant women. In addition, it may provide an easy and novel way to establish a mouse model of abortion.HighlightsCytochrome c injection induced fetal rejection.Cytochrome c injection leads to a T helper 1/T helper 2 imbalance at the maternal-fetal interface.A mouse model of abortion was established by injecting tail vein with cytochrome c.

Smart Polymersomes Dually Functionalized with cRGD and Fusogenic GALA Peptides Enable Specific and High-Efficiency Cytosolic Delivery of Apoptotic Proteins.

Low cell selectivity and uptake coupled with endosomal entrapment pose critical hurdles for intracellular delivery and clinical translation of therapeutic proteins. Herein, we report that smart polymersomes dually functionalized with cRGD and fusogenic GALA peptides (cRGD/GALA-Ps) enable ανß3-specific and high-efficiency cytosolic delivery of cytochrome C (CC), a model apoptotic protein, to A549 human lung cancer cells. cRGD/GALA-Ps was prepared with 20 mol % cRGD and varying GALA contents from 2 to 4 to 6 mol % via coassembly of PEG- b-poly(trimethylene carbonate- co-dithiolane trimethylene carbonate)-spermine (PEG- b-P(TMC- co-DTC)-spermine), cRGD-PEG- b-P(TMC- co-DTC), and maleimide-PEG- b-P(TMC- co-DTC) and postmodification using GALA-SH (sequence: CWEAALAEALAEALAEHLAEALAEALEALAA). cRGD/GALA-Ps loaded with ∼13 wt % CC displayed a small size of about 65 nm and fast glutathione-triggered protein release. Interestingly, cRGD/GALA-Ps maintained a similar targeting ability to cRGD-Ps in ανß3-positive A549 lung cancer cells, while markedly enhanced cytosolic release of FITC-labeled CC, as revealed by confocal microscopy. MTT assays exhibited that CC-loaded cRGD/GALA-Ps was significantly more potent than CC-loaded cRGD-Ps, in which cell viabilities of 76.2, 51.0, 29.6, and 35.5% were discerned for cRGD/GALA-Ps with 0, 2, 4, and 6 mol % GALA, respectively, at 15.4 µM CC. Apoptosis assays corroborated that cRGD/GALA-Ps-CC with 4 mol % GALA induced better apoptosis of A549 cells than cRGD-Ps-CC (cell apoptosis: 36.4 vs 14.4%). These results highlight that dual-functionalization of polymersomes with targeting and fusogenic peptides provides an appealing strategy for cytosolic protein delivery.

Selective staining and eradication of cancer cells by protein-carrying DARPin-functionalized liposomes.

Since their discovery, liposomes have been widely employed in biomedical research. These nano-size spherical vesicles consisting one or few phospholipid bilayers surrounding an aqueous core are capable of carrying a wide variety of bioactive compounds, including drugs, peptides, nucleic acids, proteins and others. Despite considerable success achieved in synthesis of liposome constructs containing bioactive compounds, preparation of ligand-targeted liposomes comprising large quantities of encapsulated proteins that are capable of affecting pathological cells still remains a big challenge. Here we described a novel method for preparation of small (80-90 nm in diameter) unilamellar liposomes containing very large quantities (thousands of protein molecules per liposome) of heme-containing cytochrome c, highly fluorescent mCherry and highly toxic PE40 (Pseudomonas aeruginosa Exotoxin A domain). Efficient encapsulation of the proteins was achieved through electrostatic interaction between positively charged proteins (at pH lower than pI) and negatively charged liposome membrane. The proteoliposomes containing large quantities of mCherry or PE40 and functionalized with designed ankyrin repeat protein (DARPin)_9-29, which targets human epidermal growth factor receptor 2 (HER2) were shown to specifically stain and kill in sub-nanomolar concentrations HER2-positive cells, overexpressing HER2, respectively. Specific staining and eradication of the receptor-positive cells demonstrated here makes the DARPin-functionalized liposomes carrying large quantities of fluorescent and/or toxic proteins a promising candidate for tumor detection and therapy.

Semi-interpenetrated, dendritic, dual-responsive nanogels with cytochrome c corona induce controlled apoptosis in HeLa cells.

The use of thermoresponsive nanogels (NGs) allows the controlled release of therapeutic molecules upon a thermal switch. Usually, this strategy involves the use of temperature increase to activate cargo expulsion from shrinking NGs. In this study, poly(N-isopropylacrylamide) (pNIPAM)-based NGs were involved in the release of a therapeutic protein corona by temperature decrease. NGs based on dendritic polyglycerol (dPG) and thermoresponsive pNIPAM were semi-interpenetrated with poly(4-acryloylamine-4-(carboxyethyl)heptanodioic acid) (pABC). The resulting semi-interpenetrated NGs retain the thermoresponsive properties of pNIPAM, together with pH-responsive, dendritic pABC as a secondary network, in one single nanoparticle. Semi-interpenetrated polymer network (SIPN) NGs are stable in physiological conditions, exhibit a reversible phase transition at 35 °C, together with tunable electrophoretic mobilities around the body temperature. The binding of cytochrome c (cyt c) was successful on SIPN NGs in their collapsed state at 37 °C. Upon cooling of the samples to room temperature, the swelling of the NG effectively boosted the release of cyt c, as compared with the same kept at constant 37 °C. These responsive SIPN NGs were able to deliver cyt c to cancer cells and specifically induce apoptosis at 30 °C, while the cells remained largely unaffected at 37 °C. In this way, we show therapeutic efficacy of thermoresponsive NGs as protein carriers and their efficacy triggered by temperature decrease. We envision the use of such thermal trigger as relevant for the treatment of superficial tumors, in which induction of apoptosis can be controlled by the application of local cooling agents.

An autonomous tumor-targeted nanoprodrug for reactive oxygen species-activatable dual-cytochrome c/doxorubicin antitumor therapy.

The precise tumor cell-specific delivery of therapeutic proteins and the elimination of side effects associated with routine chemotherapeutic agents are two current critical considerations for tumor therapy. In this study, we report a reactive oxygen species (ROS)-activated yolk-shell nanoplatform for the tumor-specific co-delivery of cytochrome c (Cyt c) prodrug and doxorubicin, in which the bioactivity of Cyt c could be restored by the intracellular ROS-trigger and readily initiate the sequential doxorubicin release. The DOX-loaded lactobionic acid-modified yolk-shell mesoporous silica nanoparticles were first encapsulated with 4-nitrophenyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl carbonate (NBC)-modified Cyt c via boronic ester linkages, and functionalized again with lactobionic acid to further shield Cyt c and confer the selective tumor targeting against liver cancer cells. The key feature in this design is that by taking advantage of the boronic ester linkage, the cytotoxicity of Cyt c capped on the nanoparticle could be temporarily deactivated during blood transportation and rapidly restored upon exposure to the ROS-rich microenvironment within liver cancer cells, thereby simultaneously achieving the protein therapy and stimuli-responsive doxorubicin release. This study presents a novel strategy for the development of tumor-sensitive co-delivery nanoplatforms.

Efficacious delivery of protein drugs to prostate cancer cells by PSMA-targeted pH-responsive chimaeric polymersomes.

Protein drugs as one of the most potent biotherapeutics have a tremendous potential in cancer therapy. Their application is, nevertheless, restricted by absence of efficacious, biocompatible, and cancer-targeting nanosystems. In this paper, we report that 2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic acid (Acupa)-decorated pH-responsive chimaeric polymersomes (Acupa-CPs) efficiently deliver therapeutic proteins into prostate cancer cells. Acupa-CPs had a unimodal distribution with average sizes ranging from 157-175 nm depending on amounts of Acupa. They displayed highly efficient loading of both model proteins, bovine serum albumin (BSA) and cytochrome C (CC), affording high protein loading contents of 9.1-24.5 wt.%. The in vitro release results showed that protein release was markedly accelerated at mildly acidic pH due to the hydrolysis of acetal bonds in the vesicular membrane. CLSM and MTT studies demonstrated that CC-loaded Acupa10-CPs mediated efficient delivery of protein drugs into PSMA positive LNCaP cells leading to pronounced antitumor effect, in contrast to their non-targeting counterparts and free CC. Remarkably, granzyme B (GrB)-loaded Acupa10-CPs caused effective apoptosis of LNCaP cells with a low half-maximal inhibitory concentration (IC50) of 1.6 nM. Flow cytometry and CLSM studies using MitoCapture™ revealed obvious depletion of mitochondria membrane potential in LNCaP cells treated with GrB-loaded Acupa10-CPs. The preliminary in vivo experiments showed that Acupa-CPs had a long circulation time with an elimination phase half-life of 3.3h in nude mice. PSMA-targeted, pH-responsive, and chimaeric polymersomes have appeared as efficient protein nanocarriers for targeted prostate cancer therapy.

Calcium Carbonate Mineralized Nanoparticles as an Intracellular Transporter of Cytochrome†c for Cancer Therapy.

A new intracellular delivery system based on an apoptotic protein-loaded calcium carbonate (CaCO3 ) mineralized nanoparticle (MNP) is described. Apoptosis-inducing cytochrome c (Cyt c) loaded CaCO3 MNPs (Cyt c MNPs) were prepared by block copolymer mediated in situ CaCO3 mineralization in the presence of Cyt c. The resulting Cyt c MNPs had a vaterite polymorph of CaCO3 with a mean hydrodynamic diameter of 360.5 nm and exhibited 60% efficiency for Cyt c loading. The Cyt c MNPs were stable at physiological pH (pH 7.4) and effectively prohibited the release of Cyt c, whereas, at intracellular endosomal pH (pH 5.0), Cyt c release was facilitated. The MNPs enable the endosomal escape of Cyt c for effective localization of Cyt c in the cytosols of MCF-7 cells. Flow cytometry showed that the Cyt c MNPs effectively induced apoptosis of MCF-7 cells. These findings indicate that the CaCO3 MNPs can meet the prerequisites for delivery of cell-impermeable therapeutic proteins for cancer therapy.

Mesoporous silica-layered biopolymer hybrid nanofibrous scaffold: a novel nanobiomatrix platform for therapeutics delivery and bone regeneration.

Nanoscale scaffolds that characterize high bioactivity and the ability to deliver biomolecules provide a 3D microenvironment that controls and stimulates desired cellular responses and subsequent tissue reaction. Herein novel nanofibrous hybrid scaffolds of polycaprolactone shelled with mesoporous silica (PCL@MS) were developed. In this hybrid system, the silica shell provides an active biointerface, while the 3D nanoscale fibrous structure provides cell-stimulating matrix cues suitable for bone regeneration. The electrospun PCL nanofibers were coated with MS at controlled thicknesses via a sol-gel approach. The MS shell improved surface wettability and ionic reactions, involving substantial formation of bone-like mineral apatite in body-simulated medium. The MS-layered hybrid nanofibers showed a significant improvement in mechanical properties, in terms of both tensile strength and elastic modulus, as well as in nanomechanical surface behavior, which is favorable for hard tissue repair. Attachment, growth, and proliferation of rat mesenchymal stem cells were significantly improved on the hybrid scaffolds, and their osteogenic differentiation and subsequent mineralization were highly up-regulated by the hybrid scaffolds. Furthermore, the mesoporous surface of the hybrid scaffolds enabled the loading of a series of bioactive molecules, including small drugs and proteins at high levels. The release of these molecules was sustainable over a long-term period, indicating the capability of the hybrid scaffolds to deliver therapeutic molecules. Taken together, the multifunctional hybrid nanofibrous scaffolds are considered to be promising therapeutic platforms for stimulating stem cells and for the repair and regeneration of bone.

Intracellular delivery of cytochrome c by galactosylated albumin to hepatocarcinoma cells.

In some cancer cells, translocation of cytochrome c (Cyt c) from mitochondria to the cytoplasma is inhibited. This inhibition prevents cells from undergoing apoptotic cell death and can lead to uncontrolled cell growth. Increasing cytoplasmic concentration of Cyt c can induce apoptosis in cancer cells as a strategy of cancer therapy. Here we proposed a galactosylated albumin based carrier for intracellular delivery of Cyt c to hepatocarcinoma cells. Galactosylated albumin is recognized by highly expressed asialoglycoprotein receptors (ASGPR) on hepatocarcinoma cells and is further internalized into cells via receptor mediated endocytosis. Cyt c was chemically conjugated to galactosylated albumin with a reducible disulfide linker in order to release Cyt c from the carrier inside cells. We tested cellular uptake and cytotoxicity of Cyt c conjugates in ASGPR positive and negative hepatocarcinoma cells. The results showed galatosylated albumin significantly increased cellular uptake in both cell types resulting in cytotoxicity in a dose dependent manner through the induction of apoptosis. The lack of ASGPR specific uptake might be due to other carbohydrate-recognizing receptors expressed on tumor cells. In general, our work has shown that intracellular delivery of Cyt c to tumor cells can be an alternative therapeutic approach and galactosylated albumin can be a protein drug carrier for intracellular delivery.

Delivery of chemically glycosylated cytochrome c immobilized in mesoporous silica nanoparticles induces apoptosis in HeLa cancer cells.

Cytochrome c (Cyt c) is a small mitochondrial heme protein involved in the intrinsic apoptotic pathway. Once Cyt c is released into the cytosol, the caspase mediated apoptosis cascade is activated resulting in programmed cell death. Herein, we explore the covalent immobilization of Cyt c into mesoporous silica nanoparticles (MSN) to generate a smart delivery system for intracellular drug delivery to cancer cells aiming at affording subsequent cell death. Cyt c was modified with sulfosuccinimidyl-6-[3'-(2-pyridyldithio)-propionamido] hexanoate (SPDP) and incorporated into SH-functionalized MSN by thiol-disulfide interchange. Unfortunately, the delivery of Cyt c from the MSN was not efficient in inducing apoptosis in human cervical cancer HeLa cells. We tested whether chemical Cyt c glycosylation could be useful in overcoming the efficacy problems by potentially improving Cyt c thermodynamic stability and reducing proteolytic degradation. Cyt c lysine residues were modified with lactose at a lactose-to-protein molar ratio of 3.7 ± 0.9 using mono(lactosylamido)-mono(succinimidyl) suberate linker chemistry. Circular dichroism (CD) spectra demonstrated that part of the activity loss of Cyt c was due to conformational changes upon its modification with the SPDP linker. These conformational changes were prevented in the glycoconjugate. In agreement with the unfolding of Cyt c by the linker, a proteolytic assay demonstrated that the Cyt c-SPDP conjugate was more susceptible to proteolysis than Cyt c. Attachment of the four lactose molecules reversed this increased susceptibility and protected Cyt c from proteolytic degradation. Furthermore, a cell-free caspase-3 assay revealed 47% and 87% of relative caspase activation by Cyt c-SPDP and the Cyt c-lactose bioconjugate, respectively, when compared to Cyt c. This again demonstrates the efficiency of the glycosylation to improve maintaining Cyt c structure and thus function. To test for cytotoxicity, HeLa cells were incubated with Cyt c loaded MSN at different Cyt c concentrations (12.5, 25.0, and 37.5 µg/mL) for 24-72 h and cellular metabolic activity determined by a cell proliferation assay. While MSN-SPDP-Cyt c did not induced cell death, the Cyt c-lactose bioconjugate induced significant cell death after 72 h, reducing HeLa cell viability to 67% and 45% at the 25 µg/mL and 37.5 µg/mL concentrations, respectively. Confocal microscopy confirmed that the MSN immobilized Cyt c-lactose bioconjugate was internalized by HeLa cells and that the bioconjugate was capable of endosomal escape. The results clearly demonstrate that chemical glycosylation stabilized Cyt c upon formulation of a smart drug delivery system and upon delivery into cancer cells and highlight the general potential of chemical protein glycosylation to improve the stability of protein drugs.

Efficient delivery of p53 and cytochrome c by supramolecular assembly of a dendritic multi-domain delivery system.

Versatile nanocarrier systems facilitating uptake of exogenous proteins are highly alluring in evaluating these proteins for therapeutic applications. The self-assembly of an efficient nano-sized protein transporter consisting of three different entities is presented: A streptavidin protein core functioning as an adapter, second generation polyamidoamine dendrons for facilitating cell uptake as well as two different therapeutic proteins (tumor suppressor p53 or pro-apoptotic cytochrome c as cargo). Well-defined dendrons containing a biotin core are prepared and display no cytotoxic behavior upon conjugation to streptavidin. The integration of biotinylated human recombinant p53 (B-p53) into the three component system allows excellent internalization into HeLa, A549 and SaOS osteosarcoma cells monitored via confocal microscopy, immunoblot analysis and co-localization studies. In addition, the conjugation of B-p53 to dendronized streptavidin preserves its specific DNA-binding in vitro, and its delivery into SaOS cells impairs cell viability with concomitant activation of caspases 3 and 7. The versatility of this system is further exhibited by the significant enhancement of the pro-apoptotic effects of internalized cytochrome c which is analyzed by flow cytometry and cell viability assays. These results demonstrate that the "bio-click" self-assembly of biotinylated dendrons and proteins on a streptavidin adapter yields a stable supramolecular complex. This efficient bionanotransporter provides an attractive platform for mediating the delivery of functional proteins of interest into living mammalian cells in a facile and rapid way.

Controlled intra- and transdermal protein delivery using a minimally invasive Erbium:YAG fractional laser ablation technology.

The aim of the study was (i) to investigate the feasibility of using fractional laser ablation to create micropore arrays in order to deliver proteins into and across the skin and (ii) to demonstrate how transport rates could be controlled by variation of poration and formulation conditions. Four proteins with very different structures and properties were investigated - equine heart cytochrome c (Cyt c; 12.4 kDa), recombinant human growth hormone expressed in Escherichia coli (hGH; 22 kDa), urinary follicle stimulating hormone (FSH; 30 kDa) and FITC-labelled bovine serum albumin (FITC-BSA; 70 kDa). The transport experiments were performed using a scanning Er:YAG diode pumped laser (P.L.E.A.S.E.®; Precise Laser Epidermal System). The distribution of FITC-BSA in the micropores following P.L.E.A.S.E.® poration was visualised by using confocal laser scanning microscopy (CLSM). Porcine skin was used for the device parameter and CLSM studies; its validity as a model was confirmed by subsequent comparison with transport of Cyt c and FITC-BSA across P.L.E.A.S.E.® porated human skin. No protein transport (deposition or permeation) was observed across intact skin; however, P.L.E.A.S.E.® poration enabled total delivery after 24h of 48.2±8.9, 8.1±4.2, 0.2±0.1 and 273.3±30.6 µg/cm(2) for Cyt c, hGH, FSH and FITC-BSA, respectively, using 900 pores/135.9 cm(2). Calculation of permeability coefficients showed that there was no linear dependence of transport on molecular weight ((1.6±0.3), (0.1±0.05), (0.08±0.03) and (0.9±0.1)×10(-3) cm/h, for Cyt c, hGH, FSH and FITC-BSA, respectively); indeed, a U-shaped curve was observed. This suggested that molecular weight was not a sufficiently sensitive descriptor and that transport was more likely to be determined by the surface properties of the respective proteins since these would govern interactions with the local microenvironment. Increasing pore density (i.e. the number of micropores per unit area) had a statistically significant effect on the cumulative permeation of both Cyt c (at 100, 150, 300 and 600 pores/cm(2), permeation was 11.2±2.4, 15.3±11.8, 33.8±10.5 and 51.2±15.8 4 µg/cm(2), respectively) and FITC-BSA (at 50, 100, 150 and 300 pores/cm(2), it was 58.5±15.3, 132.6±40.0, 192.7±24.4, 293.3±76.5 µg/cm(2), respectively). Linear relationships were established in both cases. However, only the delivery of FITC-BSA was improved upon increasing fluence (53.3±22.5, 293.3±76.5, 329.6±11.5 and 222.1±29.4 µg/cm(2) at 22.65, 45.3, 90.6 and 135.9 J/cm(2), respectively). The impact of fluence - and hence pore depth - on transport will depend on the relative diffusivities of the protein in the micropore and in the 'bulk' epidermis/dermis. Experiments with Cyt c and FSH confirmed that delivery was dependent upon concentration, and it was shown that therapeutic delivery of the latter was feasible. Cumulative permeation of Cyt c and FITC-BSA was also shown to be statistically equivalent across porcine and human skin. In conclusion, it was demonstrated that laser microporation enabled protein delivery into and across the skin and that this could be modulated via the poration parameters and was also dependent upon the concentration gradient in the pore. However, the role of protein physicochemical properties and their influence on transport rates remains to be elucidated and will be explored in future studies.

Biodegradable polymersomes with an ionizable membrane: facile preparation, superior protein loading, and endosomal pH-responsive protein release.

Novel biodegradable polymersomes containing an ionizable membrane were developed for efficient loading and rapid intracellular release of proteins. The polymersomes were prepared from poly(ethylene glycol)-b-poly(trimethylene carbonate) (PEG-PTMC) block copolymer derivatives containing acrylate, carboxylic acid, and amine groups along PTMC block, which are denoted as PEG-PTMC(AC), PEG-PTMC(COOH), and PEG-PTMC(NH(2)), respectively. Notably, nano-sized polymersomes (95.1-111.6nm) were formed by directly dispersing these copolymers in phosphate buffer at room temperature. Both FITC-labeled bovine serum albumin (FITC-BSA) and cytochrome C (FITC-CC) were readily loaded into PEG-PTMC(COOH) and PEG-PTMC(NH(2)) polymersomes with remarkably high loading levels. Interestingly, in vitro release studies showed that PEG-PTMC(COOH) and PEG-PTMC(NH(2)) polymersomes had pH-responsive protein release behaviors in which significantly faster protein release was observed at endosomal pH than at physiological pH. MTT assays indicated that these polymersomes had low cytotoxicity. Furthermore, confocal laser scanning microscope (CLSM) observations revealed that FITC-CC loaded polymersomes efficiently delivered proteins into MCF-7 cells following 24h incubation. Importantly, flow cytometry showed that CC-loaded polymersomes induced markedly enhanced apoptosis in MCF-7 cells as compared to free CC. These novel membrane ionizable biodegradable polymersomes have appeared as highly promising nanocarriers for efficient intracellular protein delivery.

A novel double-coating carrier produced by solid-in-oil and solid-in-water nanodispersion technology for delivery of genes and proteins into cells.

A novel intracellular delivery method both for genes and proteins is one of the most coveted systems in the drug delivery field. In the present study, we developed a double-coating carrier loaded with gene and protein produced by solid-in-oil and solid-in-water nanodispersion technology. The double-coating carriers did not require electrostatic interactions during the preparation so were able to encapsulate plasmid DNA, ovalbumin (pI 4.5), horseradish peroxidase (pI 7.2), and cytochrome-c (pI 10.5) in a consistent manner. The carriers had practical encapsulation efficiencies and release profiles for genes and proteins. Furthermore, effective gene expression and cellular uptakes of both anionic and cationic proteins were achieved by modification of carriers with functional molecules. These findings indicate that the double-coating carrier has high potential for cellular delivery of various drugs and is a novel, superior method for both gene and protein delivery into cells.

[Hepatoprotective properties of cytochrome C in traumatic toxicosis].

Experiments on rats showed that traumatic toxicosis (crush syndrome) was accompanied by disorders of both excretion and detoxication functions of the liver and a decrease in the energy potential of the liver. Systemic administration of cytochrome C (10 mg/kg) immediately after trauma and decompression increased the level of endogenous cytochrome C, recovered the pool of adenine nucleotides, normalized bromsulfaleine excretion from the blood, and decreased the content of toxic metabolites in the blood. The obtained experimental data show that cytochrome C possesses high hepatoprotective properties with respect to the development of traumatic toxicosis.

Langerin+ CD8alpha+ dendritic cells are critical for cross-priming and IL-12 production in response to systemic antigens.

Distinct dendritic cell (DC) subsets differ with respect to pathways of Ag uptake and intracellular routing to MHC class I or MHC class II molecules. Murine studies suggest a specialized role for CD8alpha(+) DC in cross-presentation, where exogenous Ags are presented on MHC class I molecules to CD8(+) T cells, while CD8alpha(-) DC are more likely to present extracellular Ags on MHC class II molecules to CD4(+) T cells. As a proportion of CD8alpha(+) DC have been shown to express langerin (CD207), we investigated the role of langerin(+)CD8alpha(+) DC in presenting Ag and priming T cell responses to soluble Ags. When splenic DC populations were sorted from animals administered protein i.v., the ability to cross-present Ag was restricted to the langerin(+) compartment of the CD8alpha(+) DC population. The langerin(+)CD8alpha(+) DC population was also susceptible to depletion following administration of cytochrome c, which is known to trigger apoptosis if diverted to the cytosol. Cross-priming of CTL in the presence of the adjuvant activity of the TLR2 ligand N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-Cys-[S]-Serl-[S]-Lys4-trihydrochloride or the invariant NKT cell ligand alpha-galactosylceramide was severely impaired in animals selectively depleted of langerin(+) cells in vivo. The production of IL-12p40 in response to these systemic activation stimuli was restricted to langerin(+)CD8alpha(+) DC, and the release of IL-12p70 into the serum following invariant NKT cell activation was ablated in the absence of langerin(+) cells. These data suggest a critical role for the langerin(+) compartment of the CD8alpha(+) DC population in cross-priming and IL-12 production.

Freeze-dried liposomes as potential carriers for ocular administration of cytochrome c against selenite cataract formation.

OBJECTIVES: In this study, the preparation, stability and anti-cataract effect of cationic freeze-dried liposomes containing cytochrome c, along with nicotinamide and adenosine, are described. METHODS: Cytochrome c-loaded cationic liposomes (CC-L) were prepared by the thin-layer evaporation technique and lyophilized to obtain freeze-dried cytochrome c liposomes (CC-F). The influence of the preparation components on the liposomal encapsulation efficiency and the stability were studied. The anti-cataract effect of the CC-F was demonstrated through attenuating lens opacity development with slit lamp examination in rats with selenite-induced cataract. KEY FINDINGS: Our study indicates that: (1) the liposomal encapsulation efficiency increased with increasing phosphatidylcholine content and reduced in the presence of stearylamine. Moreover, optimal encapsulation efficiency was obtained at an appropriate ratio of phosphatidylcholine to cholesterol; (2) CC-F was stable for at least 12 months at 4 degrees C; (3) satisfactory improvements in lens opacity were shown in the cytochrome c-treated groups, especially for the CC-F-treated group with the decreased percentage of lens opacity at about 28% at the final examination. CONCLUSIONS: CC-F were shown to be stable superior ophthalmic carriers and were able to markedly retard the onset of cataract development.

Controlled release of substances bound to fibrin-anchors or of DNA.

Fibrin sealants have been proposed as depot matrices for substances due to their biocompatibility, advantageous biological properties, and widespread use in wound healing. This study showed possibilities for a continuous and controlled release of pharmaceutically active substances out of a fibrin matrix. Substances of interest were linked to naturally occuring fibrin-anchors, (i) thrombin, (ii) fibronectin, and (iii) DNA. Fibronectin and thrombin bind fibrin by a specific binding moiety and DNA by charge. Fibrin clots were prepared from Tisseel Fibrin Sealant (Baxter AG, Vienna) by mixing 100 mg/ml fibrinogen, the substance of interest, and 4 U/ml of thrombin. Chemical crosslinking of proteins was performed with EDC using standard reaction conditions. Modification of proteins with biotin and PPACK was performed with N-hydroxysuccinimid activated compounds. With fibrin-anchors pharmaceutically active substances, i.e., tumor necrosis factor (TNF), albumin, and plasmid-DNA, were continously released over 10 days. In conclusion, the naturally occuring proteins fibronectin and thrombin with a fibrin binding moiety or DNA can be used as fibrin-anchors.

Charge-conversional polyionic complex micelles-efficient nanocarriers for protein delivery into cytoplasm.

Special delivery! Polyionic complex (PIC) micelles that contain the charge-conversional moieties citaconic amide or cis-aconitic amide were developed for cytoplasmic protein delivery. The increase of the charge density on the protein cargo helped the stability of the PIC micelles without cross-linking, and the charge-conversion in endosomes induced the dissociation of the PIC micelles to result in efficient endosomal release (see picture).