Differential proteomics analysis of the surface heterogeneity of dextran iron oxide nanoparticles and the implications for their in vivo clearance

In order to understand the role of plasma proteins in the rapid liver clearance of dextran-coated superparamagnetic iron oxide (SPIO) in vivo, we analyzed the full repertoire of SPIO-binding blood proteins using novel two-dimensional differential mass spectrometry approach. The identified proteins showed specificity for surface domains of the nanoparticles: mannan-binding lectins bound to the dextran coating, histidine-rich glycoprotein and kininogen bound to the iron oxide part, and the complement lectin and contact clotting factors were secondary binders. Nanoparticle clearance studies in knockout mice suggested that these proteins, as well as several previously identified opsonins, do not play a significant role in the SPIO clearance. However, both the dextran coat and the iron oxide core remained accessible to specific probes after incubation of SPIO in plasma, suggesting that the nanoparticle surface could be available for recognition by macrophages, regardless of protein coating. These data provide guidance to rational design of bioinert, long-circulating nanoparticles.National Cancer Institute (U.S.) (Grant CA119335)National Cancer Institute (U.S.) (Grant CA124427

Downregulation of hepatic lipopolysaccharide binding protein improves lipogenesis-induced liver lipid accumulation

Circulating lipopolysaccharide-binding protein (LBP) is increased in individuals with liver steatosis. We aimed to evaluate the possible impact of liver LBP downregulation using lipid nanoparticle-containing chemically modified LBP small interfering RNA (siRNA) (LNP-Lbp UNA-siRNA) on the development of fatty liver. Weekly LNP-Lbp UNA-siRNA was administered to mice fed a standard chow diet, a high-fat and high-sucrose diet, and a methionine- and choline-deficient diet (MCD). In mice fed a high-fat and high-sucrose diet, which displayed induced liver lipogenesis, LBP downregulation led to reduced liver lipid accumulation, lipogenesis (mainly stearoyl-coenzyme A desaturase 1 [Scd1]) and lipid peroxidation-associated oxidative stress markers. LNP-Lbp UNA-siRNA also resulted in significantly decreased blood glucose levels during an insulin tolerance test. In mice fed a standard chow diet or an MCD, in which liver lipogenesis was not induced or was inhibited (especially Scd1 mRNA), liver LBP downregulation did not impact on liver steatosis. The link between hepatocyte LBP and lipogenesis was further confirmed in palmitate-treated Hepa1-6 cells, in primary human hepatocytes, and in subjects with morbid obesity. Altogether, these data indicate that siRNA against liver Lbp mRNA constitutes a potential target therapy for obesity-associated fatty liver through the modulation of hepatic Scd1

Metastasis of Tumor Cells Is Enhanced by Downregulation of Bit1

Resistance to anoikis, which is defined as apoptosis induced by loss of integrin-mediated cell attachment to the extracellular matrix, is a determinant of tumor progression and metastasis. We have previously identified the mitochondrial Bit1 (Bcl-2 inhibitor of transcription) protein as a novel anoikis effector whose apoptotic function is independent from caspases and is uniquely controlled by integrins. In this report, we examined the possibility that Bit1 is suppressed during tumor progression and that Bit1 downregulation may play a role in tumor metastasis.Using a human breast tumor tissue array, we found that Bit1 expression is suppressed in a significant fraction of advanced stages of breast cancer. Targeted disruption of Bit1 via shRNA technology in lowly aggressive MCF7 cells conferred enhanced anoikis resistance, adhesive and migratory potential, which correlated with an increase in active Extracellular kinase regulated (Erk) levels and a decrease in Erk-directed phosphatase activity. These pro-metastasis phenotypes were also observed following downregulation of endogenous Bit1 in Hela and B16F1 cancer cell lines. The enhanced migratory and adhesive potential of Bit1 knockdown cells is in part dependent on their high level of Erk activation since down-regulating Erk in these cells attenuated their enhanced motility and adhesive properties. The Bit1 knockdown pools also showed a statistically highly significant increase in experimental lung metastasis, with no differences in tumor growth relative to control clones in vivo using a BALB/c nude mouse model system. Importantly, the pulmonary metastases of Bit1 knockdown cells exhibited increased phospho-Erk staining.These findings indicate that downregulation of Bit1 conferred cancer cells with enhanced anoikis resistance, adhesive and migratory properties in vitro and specifically potentiated tumor metastasis in vivo. These results underscore the therapeutic importance of restoring Bit1 expression in cancer cells to circumvent metastasis at least in part through inhibition of the Erk pathway

Cationic liposomes as non-viral carriers of gene medicines: resolved issues, open questions, and future promises

The clinical success of gene therapy is critically dependent on the development of efficient and safe gene delivery reagents, popularly known as "transfection vectors." The transfection vectors commonly used in gene therapy are mainly of two types: viral and non-viral. The efficiencies of viral transfection vectors are, in general, superior to their non-viral counterparts. However, the myriads of potentially adverse immunogenic aftermaths associated with the use of viral vectors are increasingly making the non-viral gene delivery reagents as the vectors of choice. Among the existing arsenal of non-viral gene delivery reagents, the distinct advantages associated with the use of cationic transfection lipids include their: (a) robust manufacture; (b) ease in handling and preparation techniques; (c) ability to inject large lipid:DNA complexes; and (d) low immunogenic response. The present review highlights the major achievements in the area of designing efficacious cationic transfection lipids, some of the more recent advances in the field of cationic liposomes-mediated gene transfer and targeted gene delivery, some unresolved issues and challenges in liposomal gene delivery, and future promises of cationic liposomes as gene-carriers in non-viral gene therapy

Design, syntheses and in vitro gene delivery efficacies of novel mono-, di- and trilysinated cationic lipids: a structure-activity investigation

Structure-activity investigation including design, syntheses, and evaluation of relative in vitro gene delivery efficacies of a novel series of cationic amphiphiles (1-10) containing mono-, di-, and trilysine headgroups are described in CHO, COS-1, and HepG2 cells. Several interesting and rather unexpected transfection profiles were observed. In general, lipid 1 with the myristyl tail used in combination with DOPE as colipid exhibited superior transfection properties compared to (a) the monolysinated analogues with longer hydrocarbon tails (lipids 2-4), (b) the dilysine (lipids 5-7) and the trilysine headgroup analogues (lipids 8-10), and (c) commercially available LipofectAmine with multiple positive charges in its polar region. As a preliminary estimate of the relative DNA-compacting efficacies of these new lysinated cationic lipids, the hydrodynamic diameters of representative lipoplexes were measured using dynamic laser light scattering technique. Our lipoplex size data are consistent with the notion that covalent grafting of an increasing number of positively charged functional groups in the headgroup region of cationic lipids need not necessarily result in more compacted lipoplexes. Both gel retardation and DNase I sensitivity assays indicated similar lipid/DNA binding interactions for all the novel mono-, di-, and trilysinated cationic lipids. MTT-assay-based cell viability results clearly demonstrate that the overall lower transfection properties of trilysine analogues (8-10) compared to their mono- (1-4) and dilysinated (5-7) counterparts are unlikely to originate from differential toxicity related effects. Taken together, the present findings support the notion that caution needs to be exercised in ensuring enhanced gene delivery efficacies of cationic lipids through covalent grafting of multiple lysine functionalities in the headgroup region

In vitro gene transfer efficacies and serum compatibility profiles of novel mono-, di-, and tri-histidinylated cationic transfection lipids: a structure-activity investigation

Recently, we demonstrated that covalent grafting of an endosome-disrupting single histidine functionality in the headgroup region imparts high gene transfer properties to cationic amphiphiles (Kumar, V. V., et al. Gene Ther. 2003, 10, 1206-1215). However, whether covalent attachment of multiple histidine functionalities in the headgroup region are capable of further enhancing the gene transfer efficacies of cationic amphiphiles remains to be explored. To this end, herein, we report on the design, syntheses, physicochemical characterizations, in vitro gene transfer properties, and serum compatibilites of three novel nontoxic cationic transfection amphiphiles containing mono-, di-, and tri-histidine functionalities in their headgroup regions (lipids 1-3) in multiple cultured cells. Significantly, findings in both the reporter gene expression assay and the whole cell histochemical X-gal staining assay support the notion that there is no linear correlation between the in vitro transfection efficacies and the number of histidine functionalities in the polar headgroup regions for histidinylated cationic amphiphiles. The relative gene transfer efficiencies, as well as the serum compatibilities, of the present histidinylated cationic amphiphiles were found to be strikingly dependent on the medium of lipoplex formation. Most importantly, high serum compatibilities (up to 50% added serum) of the lipoplexes of lipids 1 and 3 make them promising nonviral transfection vectors for future systemic applications

Recommendations of the Oligonucleotide Safety Working Group’s Formulated Oligonucleotide Subcommittee for the Safety Assessment of Formulated Oligonucleotide-Based Therapeutics

The use of lipid formulations has greatly improved the ability to effectively deliver oligonucleotides and has been instrumental in the rapid expansion of therapeutic development programs employing oligonucleotide drugs. However, the development of such complex, multi-component therapeutics requires the implementation of unique, scientifically sound approaches based upon a hybrid of knowledge and experiences drawn from small-molecule, protein and oligonucleotide therapeutic drug development. The relative paucity of directly applicable regulatory guidance documents for oligonucleotide therapeutics in general has resulted in the generation of multiple white papers from oligonucleotide drug development experts and members of the Oligonucleotide Safety Working Group (OSWG). The members of the Formulated Oligonucleotide Subcommittee of the OSWG have utilized their collective experience working with a variety of formulations and their associated oligonucleotide ‘payloads’, as well as their insights into regulatory considerations and expectations, to generate a series of consensus recommendations for the pharmacokinetic characterization and nonclinical safety assessment of this unique class of therapeutics

Example of fatty acid-loaded lipoplex in enhancing in vitro gene transfer efficacies of cationic amphiphile

Herein, we report on the design and synthesis of a novel nontoxic cationic amphiphile N,N-di-n-tetradecyl-N-[2-[N',N'-bis(2-hydroxyethyl)amino]ethyl]-N-(2-hydroxyethyl)ammonium chloride (lipid 1) whose in vitro gene transfer efficacies in CHO, COS-1, MCF-7, and HepG2 cells are remarkably enhanced when used in combination with 30 mole percent added myristic acid. Reporter gene expression assay using p-CMV-SPORT-β-gal reporter gene revealed poor gene transfer properties of the cationic liposomes of lipid 1 and cholesterol (colipid). However, the in vitro gene delivery efficacies of lipid 1 were found to be remarkably enhanced when the cationic liposomes of lipid 1 and cholesterol were prepared in the presence of 30 mole percent added myristic acid (with respect to lipid 1) as the third liposomal ingredient. The whole cell histochemical X-gal staining of representative CHO cells further confirmed the significantly enhanced gene transfer properties of the fatty acid-loaded cationic liposomes of lipid 1 and cholesterol. Electrophoretic gel patterns in the gel mobility shift assay supports the notion that better DNA release from fatty acid lipoplexes might play a role in their enhanced gene transfer properties. In addition, such myristic acid-loaded lipoplexes of lipid 1 were also found to be serum-compatible up to 30% added serum. Taken together, our present findings demonstrate that the transfection efficacies of fatty acid-loaded lipoplexes are worth evaluating particularly when traditional cationic liposomes prepared with either cholesterol or DOPE colipids fail to transfect cultured cells

In vitro gene transfer efficacies of N,N-dialkylpyrrolidinium chlorides: a structure-activity investigation

Inspired by the previously reported superior gene transfer efficacies of amine headgroup-containing cationic lipids to their hydroxy counterparts, in the present structure-activity investigation we have compared the relative in vitro gene transfer efficacies of eight newly synthesized structural analogues of our previously reported lipids 1-4, namely the four 3,4-diaminopyrrolidinium chloride structural analogues (lipids 9-12, Chart 1) and the N-BOC-protected precursors of these amine analogues (lipids 5-8, Chart 1) with our previously reported lipids 1-4 (Chart 1) in five cultured cell lines. In contrast to the above-mentioned earlier reports, except for the superior or comparable transfection efficacies of the diaminopyrrolidinium lipids with distearyl and stearyloleyl chains (lipid 11 and 12 respectively, Chart 1) in MCF-7 and HEK293T cells, the relative transfection efficacies of the other diamino analogues were found to be much lower than their dihydroxy counterparts. The results of the DNase I sensitivity assays indicate that enhanced degradation of DNA associated with lipids 9-12 by cellular DNase I might play an important role behind their seriously compromised transfection efficacies. In addition, the present structure-activity investigation revealed a strikingly cell tropic transfection behavior of lipid 6 (Chart 1). While lipids 5, 7, and 8 were found to be either poor or essentially incompetent in transfecting all the five cells, lipid 6 was remarkably efficacious in transfecting kidney cells (COS-1 and HEK293T cells) at lipid:DNA charge ratios 3:1 and 1:1 when used in combination with equimolar amounts of DOPE and DOPC