Tissue-specific Delivery Research Articles

Synthesis of cytoadhesive poly(methylmethacrylate) for applications in targeted drug delivery.

The objective of this work was to incorporate cytoadhesive properties into poly(methylmethacrylate) (PMMA) for potential applications in highly localized tissue-specific drug delivery. First, the PMMA was chemically modified by aminolysis to yield amine-terminated surfaces. X-ray photoelectron microscopy confirmed the presence of surface nitrogen entities, and the distribution of amine groups was found to be relatively uniform, as characterized by atomic force microscopy. The availability of these groups for attachment of biologically active molecules was characterized by fluorescence microscopy after immobilization of avidin-FITC. To render the PMMA cytoadhesive, avidin molecules were conjugated to the amine-terminated surfaces with a hydroxy-succinimide-catalyzed carbodiimide reagent and biotin-labeled lectins (tomato, which binds selectively to Caco-2 cells, and peanut, an unrelated lectin) subsequently were attached utilizing avidin-biotin chemistry. Cytoadhesive activity was evaluated by characterizing the interactions between microfabricated PMMA particles and Caco-2 monolayers. After 15-, 30-, 60-, and 120-min incubation periods, the tomato lectin-conjugated PMMA showed a two to sixfold increase in Caco-2 cell recognition over control particles. Furthermore, the stability of the cytoadhesive PMMA interactions appeared to be three to seven times greater than that of the control surfaces. These findings demonstrate that cytoadhesive properties of modified PMMA, making this novel bioactive polymer very promising for applications in targeted drug delivery.

Ultrasound-targeted microbubble destruction can repeatedly direct highly specific plasmid expression to the heart.

Noninvasive, tissue-specific delivery of therapeutic agents would be a valuable clinical tool. We have previously shown that ultrasound-targeted microbubble destruction can direct expression of an adenoviral reporter to the heart. The present study shows that this method can be applied to selectively deliver plasmid vectors to the heart. We used albumin and lipid microbubbles containing plasmids with a luciferase transgene to target the heart in rats. After 4 days, organs were harvested and analyzed for reporter gene expression. In a second set of experiments, the hearts of rats treated with plasmids were harvested at various time points during a 4-week period. Both luciferase activity and mRNA concentrations were measured. Luciferase transfection with plasmids showed highly specific gene expression in the heart, with hardly any activity in control organs. Time course evaluation showed high transgene expression in the first 4 days, with a rapid decline thereafter. Repeated treatment produced a second peak of transgene expression with similar decay. Ultrasound-mediated destruction of microbubbles directs plasmid transgene expression to the heart with much greater specificity than viral vectors and can be regulated by repeated treatments. This noninvasive technique is a promising method for cardiac gene therapy.

Crossing the Membrane: Nonviral and Viral Delivery Methods for Use In Vitro and In Vivo

DNA and Cell BiologyVol. 21, No. 12 ArticlesCrossing the Membrane: Nonviral and Viral Delivery Methods for Use In Vitro and In VivoMarie A. BogoyevitchMarie A. BogoyevitchSearch for more papers by this authorPublished Online:6 Jul 2004https://doi.org/10.1089/104454902762053819AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail "Crossing the Membrane: Nonviral and Viral Delivery Methods for Use In Vitro and In Vivo." , 21(12), pp. 855–856FiguresReferencesRelatedDetailsCited byMicrofluidic platform for hepatitis B viral replication study29 December 2007 | Biomedical Microdevices, Vol. 10, No. 3 Volume 21Issue 12Dec 2002 To cite this article:Marie A. Bogoyevitch.Crossing the Membrane: Nonviral and Viral Delivery Methods for Use In Vitro and In Vivo.DNA and Cell Biology.Dec 2002.855-856.http://doi.org/10.1089/104454902762053819Published in Volume: 21 Issue 12: July 6, 2004PDF download

Plasmid DNA-entrapped nanoparticles engineered from microemulsion precursors: in vitro and in vivo evaluation.

Nonviral gene therapy has been a rapidly growing field. However, delivery systems that can provide protection for pDNA and potential targeting are still desired. A novel pDNA-nanoparticle delivery system was developed by entrapping hydrophobized pDNA inside nanoparticles engineered from oil-in-water (O/W) microemulsion precursors. Plasmid DNA was hydrophobized by complexing with cationic surfactants DOTAP and DDAB. Warm O/W microemulsions were prepared at 50-55 degrees C with emulsifying wax, Brij 78, Tween 20, and Tween 80. Nanoparticles were engineered by simply cooling the O/W microemulsions containing the hydrophobized pDNA in the oil phase to room temperature while stirring. The nanoparticles were characterized by particle sizing, zeta-potential, and TEM. Nanoparticles were challenged with serum nucleases to assess pDNA stability. In addition, the nanoparticles were coincubated with simulated biological media to assess their stability. In vitro hepatocyte transfection studies were completed with uncoated nanoparticles or nanoparticles coated with pullulan, a hepatocyte targeting ligand. In vivo biodistribution of the nanoparticles containing I-125 labeled pDNA was monitored 30 min after tail-vein injection to Balb/C mice. Depending on the hydrophobizing lipid agent employed, uniform pDNA-entrapped nanoparticles (100-160 nm in diameter) were engineered within minutes from warm O/W microemulsion precursors. The nanoparticles were negatively charged (-6 to -15 mV) and spherical. An anionic exchange column was used to separate unentrapped pDNA from nanoparticles. Gel permeation chromatography of pDNA-entrapped and serum-digested nanoparticles showed that the incorporation efficiency was approximately 30%. Free 'naked' pDNA was completely digested by serum nucleases while the entrapped pDNA remained intact. Moreover, in vitro transfection studies in Hep G2 cells showed that pullulan-coated nanoparticles resulted in enhanced luciferase expression, compared to both pDNA alone and uncoated nanoparticles. Preincubation of the cells with free pullulan inhibited the transfection. Finally, 30 min after tail vein injection to mice, only 16% of the 'naked' pDNA remained in the circulating blood compared to over 40% of the entrapped pDNA. Due to the apparent stability of these pDNA-entrapped nanoparticles in the blood, they may have potential for systemic gene therapy applications requiring cell and/or tissue-specific delivery.

Altering retroviral tropism using a random-display envelope library.

Tissue-specific gene delivery is an important aspect of many gene therapy applications. The experiments reported here constitute the first successful demonstration that cell-specific entry can be obtained by screening a random library of retroviral envelope proteins produced from a mammalian cell system. The library consisted of 10(6) different subgroup A feline leukemia virus envelope protein variants with 10 randomly substituted amino acids in the receptor-determining region. Selecting the library for fully functional envelope proteins able to mediate stable gene transfer resulted in the identification of a single envelope protein variant (EF). Subsequent examination of the host range of EF revealed that it was highly specific for D17 canine osteosarcoma cells. This was in contrast to the host ranges of the parental subgroup A and closely related subgroup C envelope proteins. Interference assays on D17 cells further indicated that receptor usage by EF was also altered compared with the A and C envelope proteins. The EF envelope protein thus isolated should be useful for studying gene therapy treatments of osteosarcoma in a large-animal model.

Targeting endothelium and its dynamic caveolae for tissue-specific transcytosis in vivo: a pathway to overcome cell barriers to drug and gene delivery.

Site-directed pharmacodelivery is a desirable but elusive goal. Endothelium and epithelium create formidable barriers to endogenous molecules as well as targeted therapies in vivo. Caveolae provide a possible, yet unproven, transcellular pathway to overcome such barriers. By using an antibody- and subfractionation-based strategy, we generated a monoclonal antibody specific for lung caveolae (TX3.833) that targets rat lungs after i.v. injection (up to 89% of dose in 30 min). Unlike control antibodies (nonbinding or to lipid rafts), TX3.833 targets lung caveolae that bud to form free vesicles for selective and quantal transendothelial transport to underlying tissue cells in vivo. Rapid sequential transcytosis can occur to the alveolar air space via epithelial caveolae. Conjugation to TX3.833 increases drug delivery to the lung up to 172-fold and achieves rapid, localized bioefficacy. We conclude that: (i) molecular heterogeneity of the endothelium and its caveolae permits vascular targeting to achieve theoretical expectations of tissue-specific delivery and bioefficacy; (ii) caveolae can mediate selective transcytosis in vivo; and (iii) targeting caveolae may provide a tissue-specific pathway for overcoming key cell barriers to many drug and gene therapies in vivo.

The prospect of gene therapy for prostate cancer: update on theory and status.

Molecularly based novel therapeutic agents are needed to address the problem of locally recurrent, or metastatic, advanced hormone-refractory prostate cancer. Recent basic science advances in mechanisms of gene expression, vector delivery, and targeting have rendered clinically relevant gene therapy to the prostatic fossa and distant sites feasible in the near future. Current research and clinical investigative efforts involving methods for more effective vector delivery and targeting, with enhanced gene expression to selected (specific) sites, are reviewed. These areas of research involve tissue-specific promoters, transgene exploration, vector design and delivery, and selective vector targeting. The 'vectorology' involved mainly addresses selective tissue homing with ligands, mechanisms of innate immune system evasion for durable transgene expression, and the possibility of repeat administration.

Genetic chemistry: Tools for gene therapy coming from unexpected directions

Overcoming the limitations of foreign gene delivery to eukaryotic cells is a crucial path toward human gene therapy. Over the last 5 years, this challenge has been approached in different ways. The design and synthesis of novel cationic lipids for gene delivery was a central preoccupation to obtain increased transgene expression. Significant progress was achieved for in vitro and in vivo gene delivery by designing a series of geometrically differing lipopolyamines. First, the geometry of lipopolyamines affects the final transgene expression. Second, introduction of DNA complexing peptides into lipopolyamine/DNA complexes is advantageous for transgene expression in the presence of serum. In a third approach, the introduction of different cationic entities such as polyguanidinium or polycyclic guanidinium instead of the polyamine head group may favor tissue-specific gene delivery. In a fourth approach, introduction of a reduction-sensitive group in lipopolyamines lead to increased transgene expression as compared to nonreducible lipopolyamines. Finally, the design and synthesis of novel nonelectrostatic DNA complexing agents represent a new promising approach for in vivo DNA delivery. The last exciting challenge to be faced was the site-specific chemical ligation to plasmid DNA as a tool for plasmid DNA targeting and intracellular localization of complexes. Together with the development of the different approaches, the physicochemical characterization of the different gene delivery systems, as well as their intracellular fate, were studied. These studies are of great importance for understanding the differing transfection efficiency obtained for different types of DNA complexes, thus a more rational base can be used for designing new generations of highly efficient nonviral self-assembling systems for gene delivery. Drug Dev. Res. 50:566–572, 2000. © 2000 Wiley-Liss, Inc.

Analysis of muscle creatine kinase regulatory elements in recombinant adenoviral vectors.

Adenoviral gene transfer holds promise for gene therapy, but effective transduction of a large and distributed tissue such as muscle will almost certainly require systemic delivery. In this context, the use of muscle-specific regulatory elements such as the muscle creatine kinase (MCK) promoter and enhancer will avoid potentially harmful ectopic expression of transgenes. We describe here the development and testing of adenoviral vectors containing small, striated muscle-specific, highly active MCK expression cassettes. One of these regulatory elements (CK6) is less than 600 bp in length and is 12% as active as the CMV promoter/enhancer in muscle. A recombinant adenoviral vector containing this regulatory element retains very high muscle specificity, expressing 600-fold higher levels of transgene in muscle than in liver. Muscle-specific regulatory elements may also increase persistence of transduced muscle cells. Adenoviral transduction of dendritic cells has been shown to stimulate cytotoxic T-lymphocyte (CTL) responses directed against transgene epitopes. We show that human dendritic cells infected in vitro with MCK-containing adenoviruses do not express significant levels of transgene. Furthermore, while adenoviral vectors containing nonspecific promoters are normally cleared from muscle tissue within 1 month, we show that MCK-containing vectors express significant levels of transgene 4 months after intramuscular injection.

Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium.

The noninvasive, tissue-specific delivery of therapeutic agents to the heart would be a valuable clinical tool. This study addressed the hypothesis that albumin-coated microbubbles could be used to effectively deliver an adenoviral transgene to rat myocardium by ultrasound-mediated microbubble destruction. Recombinant adenovirus containing beta-galactosidase and driven by a constitutive promoter was attached to the surface of albumin-coated, perfluoropropane-filled microbubbles. These bubbles were infused into the jugular vein of rats with or without simultaneous echocardiography. Additional controls included ultrasound of microbubbles that did not contain virus, virus alone, and virus plus ultrasound. One group underwent ultrasound-mediated destruction of microbubbles followed by adenovirus infusion. Rats were killed after 4 days and examined for beta-galactosidase expression. The hearts of all rats that underwent ultrasound-mediated destruction of microbubbles containing virus showed nuclear staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside substrate, indicating expression of the transgene. None of the control animals showed myocardial expression of the beta-galactosidase transgene. By quantitative analysis, beta-galactosidase activity was 10-fold higher in the treated group than in controls (P<0.0001). Ultrasound-mediated destruction of albumin-coated microbubbles is a promising method for the delivery of bioactive agents to the heart.

Genetic chemistry: Tools for gene therapy coming from unexpected directions

Overcoming the limitations of foreign gene delivery to eukaryotic cells is a crucial path toward human gene therapy. Over the last 5 years, this challenge has been approached in different ways. The design and synthesis of novel cationic lipids for gene delivery was a central preoccupation to obtain increased transgene expression. Significant progress was achieved for in vitro and in vivo gene delivery by designing a series of geometrically differing lipopolyamines. First, the geometry of lipopolyamines affects the final transgene expression. Second, introduction of DNA complexing peptides into lipopolyamine/DNA complexes is advantageous for transgene expression in the presence of serum. In a third approach, the introduction of different cationic entities such as polyguanidinium or polycyclic guanidinium instead of the polyamine head group may favor tissue-specific gene delivery. In a fourth approach, introduction of a reduction-sensitive group in lipopolyamines lead to increased transgene expression as compared to nonreducible lipopolyamines. Finally, the design and synthesis of novel nonelectrostatic DNA complexing agents represent a new promising approach for in vivo DNA delivery. The last exciting challenge to be faced was the site-specific chemical ligation to plasmid DNA as a tool for plasmid DNA targeting and intracellular localization of complexes. Together with the development of the different approaches, the physicochemical characterization of the different gene delivery systems, as well as their intracellular fate, were studied. These studies are of great importance for understanding the differing transfection efficiency obtained for different types of DNA complexes, thus a more rational base can be used for designing new generations of highly efficient nonviral self-assembling systems for gene delivery. Drug Dev. Res. 50:566–572, 2000. © 2000 Wiley-Liss, Inc.

Functional interactions between monomers of the retroviral envelope protein complex.

Retroviral vectors have been widely used in human gene therapy protocols. Entry into target cells is directed by the retroviral envelope protein, with receptor binding and postbinding fusion functions contributed mainly by the SU and TM subunits, respectively. We have generated mutants of the Moloney murine leukemia virus (MoMuLV) envelope protein with mutations in both the receptor binding domain of SU and throughout the TM subunit that are functionally inactive when expressed individually. However, the coexpression of these two classes of mutants partially restores envelope protein function and allows transduction. Several lines of evidence indicate that this complementation occurs in trans within envelope protein heterooligomers. The finding that the binding and postbinding functions of a retroviral envelope protein can be contributed by two different monomers should assist in the engineering of envelope proteins for tissue-specific gene delivery.

Tissue Distribution and Metabolism of the [32P]-Labeled Oligodeoxynucleoside Methylphosphonate-Neoglycopeptide Conjugate, [YEE(ah-GalNAc)3]-SMCC-AET-pUmpT7, in the Mouse

Development of oligodeoxynucleotides (oligo-dNs) and their analogs as therapeutic agents is complicated by their low rate of transport across cellular membranes, which is required for interaction with the intracellular complementary nucleic acid sequences, and the lack of tissue-specific delivery. To overcome these obstacles, bioconjugates between cell surface receptor ligands and oligodeoxynucleoside methylphosphonates (oligo-MPs) have been constructed containing homogeneous, chemically defined covalent linkages. We have previously established that a model conjugate, [32P]-labeled [YEE(ah-GalNAc)3]-SMCC-AET-pUmpT7 (1), is delivered to Hep G2 cells in a ligand-specific manner, reaching a peak value of 26 pmol per 10(6) cells after 24 hours incubation at 37 degrees C (Hangeland et al., 1995). In this work, the in vivo behavior of this conjugate is explored. Administration of this conjugate to mice via tail vein injection demonstrates rapid uptake in liver to the extent of 69.9 +/- 9.9% of the injected dose after 15 minutes. Thereafter, the conjugate and its metabolites are rapidly cleared via the kidney and urine. Polyacrylamide gel electrophoresis analysis of extracts of Hep G2 cells and mouse liver reveal the conjugate 1 to be extensively metabolized. In contrast, the conjugate found in mouse urine is largely intact. These data show that this novel, biodegradable delivery vehicle represents a viable approach for the delivery of antisense oligo-MPs and other oligo-dN analogs to the liver for therapeutic and diagnostic applications.

Prevention of adoptively transferred diabetes in nonobese diabetic mice with IL-10-transduced islet-specific Th1 lymphocytes. A gene therapy model for autoimmune diabetes.

Four pancreatic islet-specific CD4+ helper T (Th) 1 (Th1) clones and two Th1 clones transduced with an SRalpha promoter-linked murine IL-10 (mIL-10) cDNA of 2.0-6.0 x 10(6) cells were adoptively transferred to nonobese diabetic (NOD) mice at age 8 d. Cyclophosphamide (CY) was administered at age 37 d (plus CY), and the incidence of diabetes and the histological grade of insulitis were examined at age 47 d. After the adoptive transfer of IL-10-transduced Th1 cells, polymerase chain reaction (PCR) and reverse-transcription (RT)-PCR detected the neo gene and the retrovirus vector-mediated IL-10 mRNA in situ in recipient islets, respectively. RT-PCR detected the decrease of IFN-gamma mRNA relative to IL-10 mRNA in IL-10-transduced Th1 clones in vitro and also in recipient islets. All four wild type Th1 clones plus CY induced the insulitis grade of 2.75 and diabetes in 66% of recipient NOD mice. IL-10-transduced two Th1 clones plus CY induced periinsulitis with the grade of 1.43 and diabetes in 8.0%. The 1:1 mixture of wild type Th1 cells and IL-10-transduced Th1 cells plus CY induced periinsulitis with the grade of 1.85 and diabetes in 20%. The suppression of diabetes through decreasing IFN-gamma mRNA by the tissue-specific delivery of IL-10 to pancreatic islets with IL-10-transduced Th1 cells affords us the starting basis to develop the gene therapy for autoimmune diabetes.

Cell-Specific, Multidrug Delivery System Using Streptavidin–Protein A Fusion Protein

Tissue-specific delivery of variety of molecules has been a valuable technique for biological and medical research and for the diagnosis and therapy of cancer. We have therefore examined the ability of streptavidin–protein A (ST–PA) fusion protein complexed with monoclonal antibodies (mAbs) to transfer biotinylated proteins into specific type of cells. ST–PA/mAbs complexes could efficiently deliver biotinylated β-galactosidase into a variety of cancer cell lines through molecules expressed on their surface. In addition, ST–PA/mAb complexed with either biotinylated glucose oxidase or biotinylated ribonuclease A could be transferred to specific cell types and made to display cytotoxic activity against the transduced cell. The flexibility of the system was enhanced by the fact that the cell-targeting specificity could be altered by just changing the mAb used and the “payload” molecule could be replaced by substituting one biotinylated protein or enzyme with another. This flexibility was achieved without the need to generate a covalent chemical link or engineering new recombinant molecules. Results obtained to date suggest that the ST–PA fusion protein may be used as a nearly “universal carrier” to transfer a variety of effector molecules into target cells with a high degree of specificity. Essentially, the ST–PA fusion protein effectively serves as a high-efficiency, modular “molecular bridge” for the transfer into cells of a wide variety of effector molecules.

Multi-drug delivery system using streptavidin-transforming growth factor-alpha chimeric protein.

Tissue-specific delivery of a variety of molecules has been a valuable technique for biological and medical research. Therefore, we have constructed a recombinant plasmid containing the coding regions for streptavidin core and mature human transforming growth factor-alpha (TGF-alpha). The recombinant plasmid has been expressed in Escherichia coli to produce a chimeric protein with both streptavidin and TGF-alpha activity. The streptavidin-TGF-alpha chimeric protein (ST-TGF-alpha) could efficiently transfer biotinylated beta-galactosidase into A431 cells via the epidermal growth factor receptor. More than 99% of the cells contained the enzyme transferred. Furthermore, ST-TGF-alpha complexed with biotinylated-glucose oxidase had a significant cytotoxic effect when incubated with A431 cells. These findings suggest that the ST-TGF-alpha chimeric protein could be used to deliver proteins of interest into target cells without the need for chemical linkage or genetic construction. Essentially, ST-TGF-alpha serves as a high-modular "molecular bridge" for the passage of a wide variety of effector molecules into target cells.

Membrane Transporters as Determinant of Drug Absorption and Disposition.

Although passive diffusion, which depends on the lipid solubility, is a fundamental mechanism for the membrane transport of many of compounds, water-soluble compounds also cross cell membranes by the specialized carrier-mediated transport mechanisms. We have demonstrated that several drugs are transported by the tissue-specific transporters in intestinal and renal epithelial cells, hepatocytes and brain capillary endothelial cells which form the blood-brain barrier. They include oligopeptide transporter, monocarboxylic acid transporter, anion antiporter, organic anion transporter, and P-glycoprotein. Most of them functions for the uptakes of drugs into the cells leading to the increased permeability, others exclude drugs out of cells, thereby decreasing apparent permeability into cells. It was also found that a certain drug which crosses by the certain transporter in the intestinal membranes is sometimes recognized by the other transporter, or not recognized by transporters in other tissues. This finding demonstrates that it would be possible to expect tissue specific delivery of drugs by utilizing transporters which have different characteristics among tissues. Such a difference in permeability among tissues would be difficult to expect when the membrane transport is carried out by passive diffusion. There are more evidences for carrier-mediated transport of many drugs in various tissues other than mentioned in the present study. Further mechanistic clarification and quantitative analysis of pharmacokinetic importance of such transporters will help the development of effective strategies for the site specific drug delivery.

Therapeutic opportunities involving cellular oncogenes: novel approaches fostered by biotechnology.

Biotechnological processes are having a major impact on many industrial sectors, including the pharmaceutical industry. The contributions of recombinant DNA and hybridoma technologies to modern therapeutics include production of natural and unnatural peptides, subunit vaccines, monoclonal antibodies and nucleic acid hybridization probes for in vitro and in vivo diagnostics and biological imaging, therapeutic monoclonal antibodies as tissue-specific delivery systems or as agents to confer passive immunity, production of therapeutic targets for rational drug design, and the use of cloned enzymes as stereospecific catalysts in large-scale production of small medicinal molecules. Biotechnological advances have led to the identification of a discrete set of genes, oncogenes, which may be essential contributing factors for a great variety and number of human cancers. In addition, biotechnological innovations are fostering the exploitation of oncogenes as novel therapeutic targets for cancer diagnosis, prognosis, and treatment. Because oncogenes are activated in transformation by either qualitative or quantitative mechanisms, however, different biotechnology-based therapeutic approaches are required for each class.