CD8+ T cells mediate protection against Zika virus induced by an NS3-based vaccine.

Zika virus (ZIKV) is associated with congenital malformations in infants born to infected mothers, and with Guillain-Barré syndrome in infected adults. Development of ZIKV vaccines has focused predominantly on the induction of neutralizing antibodies, although a suboptimal antibody response may theoretically enhance disease severity through antibody-dependent enhancement (ADE). Here, we report induction of a protective anti-ZIKV CD8+ T cell response in the HLA-B*0702 Ifnar1-/- transgenic mice using an alphavirus-based replicon RNA vaccine expressing ZIKV nonstructural protein NS3, a potent T cell antigen. The NS3 vaccine did not induce a neutralizing antibody response but elicited polyfunctional CD8+ T cells that were necessary and sufficient for preventing death in lethally infected adult mice and fetal growth restriction in infected pregnant mice. These data identify CD8+ T cells as the major mediators of ZIKV NS3 vaccine-induced protection and suggest a new strategy to develop safe and effective anti-flavivirus vaccines.

Recognition of dextran-superparamagnetic iron oxide nanoparticle conjugates (Feridex) via macrophage scavenger receptor charged domains.

Dextran-coated superparamagnetic iron oxide nanoparticles (dextran-SPIO conjugates) offer the attractive possibility of enhancing MRI imaging sensitivity so that small or diffuse lesions can be detected. However, systemically injected SPIOs are rapidly removed by macrophages. We engineered embryonic cells (HEK293T) to express major macrophage scavenger receptor (SR) subtypes including SR-AI, MARCO, and endothelial receptor collectin-12. These SRs possess a positively charged collagen-like (CL) domain and they promoted SPIO uptake, while the charge neutral lipoprotein receptor SR-BI did not. In silico modeling indicated a positive net charge on the CL domain and a net negative charge on the cysteine-rich (CR) domain of MARCO and SR-AI. In vitro experiments revealed that CR domain deletion in SR-AI boosted uptake of SPIO 3-fold, while deletion of MARCO's CR domain abolished this uptake. These data suggest that future studies might productively focus on the validation and further exploration of SR charge fields in SPIO recognition.

Different effect of hydrogelation on antifouling and circulation properties of dextran-iron oxide nanoparticles.

Premature recognition and clearance of nanoparticulate imaging and therapeutic agents by macrophages in the tissues can dramatically reduce both the nanoparticle half-life and delivery to the diseased tissue. Grafting nanoparticles with hydrogels prevents nanoparticulate recognition by liver and spleen macrophages and greatly prolongs circulation times in vivo. Understanding the mechanisms by which hydrogels achieve this "stealth" effect has implications for the design of long-circulating nanoparticles. Thus, the role of plasma protein absorption in the hydrogel effect is not yet understood. Short-circulating dextran-coated iron oxide nanoparticles could be converted into stealth hydrogel nanoparticles by cross-linking with 1-chloro-2,3-epoxypropane. We show that hydrogelation did not affect the size, shape and zeta potential, but completely prevented the recognition and clearance by liver macrophages in vivo. Hydrogelation decreased the number of hydroxyl groups on the nanoparticle surface and reduced the binding of the anti-dextran antibody. At the same time, hydrogelation did not reduce the absorption of cationic proteins on the nanoparticle surface. Specifically, there was no effect on the binding of kininogen, histidine-rich glycoprotein, and protamine sulfate to the anionic nanoparticle surface. In addition, hydrogelation did not prevent activation of plasma kallikrein on the metal oxide surface. These data suggest that (a) a stealth hydrogel coating does not mask charge interactions with iron oxide surface and (b) the total blockade of plasma protein absorption is not required for maintaining iron oxide nanoparticles' long-circulating stealth properties. These data illustrate a novel, clinically promising property of long-circulating stealth nanoparticles.

Role of carbohydrate receptors in the macrophage uptake of dextran-coated iron oxide nanoparticles.

Superparamagnetic iron oxide (SPIO, Ferumoxides, Feridex), an important MRI intravenous contrast reagent, is efficiently recognized and eliminated by macrophages in the liver, spleen, lymph nodes and atherosclerotic lesions. The receptors that recognize nanoparticles are poorly defined and understood. Since SPIO is coated with bacterial polysaccharide dextran, it is important to know whether carbohydrate recognition plays a role in nanoparticle uptake by macrophages. Lectin-like receptors CD206 (macrophage mannose receptor) and SIGNR1 were previously shown to mediate uptake of bacterial polysaccharides. We transiently expressed receptors MGL-1, SIGNR-1 and msDectin-1 in non-macrophage 293T cells using lipofection. The expression was confirmed by reverse transcription PCR. Following incubation with the nanoparticles, the uptake in receptor-expressing cells was not statistically different compared to control cells (GFP-transfected). At the same time, expression of scavenger receptor SR-A1 increased the uptake of nanoparticles three-fold compared to GFP-transfected and control vector-transfected cells. Blocking CD206 with anti-CD206 antibody or with the ligand mannan did not affect SPIO uptake by J774.A1 macrophages. Similarly, there was no inhibition of the uptake by anti-CD11b (Mac-1 integrin) antibody. Polyanionic scavenger receptor ligands heparin, polyinosinic acid, fucoidan and dextran sulfate decreased the uptake of SPIO by J774A.1 macrophages and Kupffer cells by 60-75%. These data unambiguously show that SPIO is taken up via interaction by scavenger receptors, but not via dextran recognition by carbohydrate receptors. Understanding of nanoparticle-receptor interaction can provide guidance for the design of long circulating, non-toxic nanomedicines.

Metastasis of tumor cells is enhanced by downregulation of Bit1.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

Interactions of nanoparticles with plasma proteins: implication on clearance and toxicity of drug delivery systems.

INTRODUCTION: Intravenously injected nanoparticles, like any other foreign pathogen that enters the body, encounter multiple lines of defense intended to neutralize and eliminate the invading substance. Adsorption of plasma proteins on the nanoparticle surface is the first barrier of defense, which could lead to physical changes in the formulation, such as aggregation and charge neutralization, biochemical activation of defense cascades, and trigger elimination by multiple types of phagocytic cell. AREAS COVERED: In this review, recent knowledge on the mechanisms that govern the interactions of nanoparticles (micelles, liposomes, polymeric and inorganic nanoparticles) with plasma proteins is discussed. In particular, the role of the nanoparticle surface properties and protective polymer coating in these interactions is described. The mechanisms of protein adsorption on different nanoparticles are analyzed and the implications on the clearance, toxicity and efficacy of drug delivery are discussed. The review provides readers with the biological insight into the plasma/blood interactions of nanoparticles. EXPERT OPINION: The immune recognition of nanoparticles can seriously affect the drug delivery efficacy and toxicity. There is at present not enough knowledge on the mechanisms that dictate the nanoparticle immune recognition and stability in the biological milieu. Understanding the mechanisms of recognition will become an important part of nanoparticle design.

Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs.

Poor penetration of anticancer drugs into tumors can be an important factor limiting their efficacy. We studied mouse tumor models to show that a previously characterized tumor-penetrating peptide, iRGD, increased vascular and tissue permeability in a tumor-specific and neuropilin-1-dependent manner, allowing coadministered drugs to penetrate into extravascular tumor tissue. Importantly, this effect did not require the drugs to be chemically conjugated to the peptide. Systemic injection with iRGD improved the therapeutic index of drugs of various compositions, including a small molecule (doxorubicin), nanoparticles (nab-paclitaxel and doxorubicin liposomes), and a monoclonal antibody (trastuzumab). Thus, coadministration of iRGD may be a valuable way to enhance the efficacy of anticancer drugs while reducing their side effects, a primary goal of cancer therapy research.

Tissue-penetrating delivery of compounds and nanoparticles into tumors.

Poor penetration of drugs into tumors is a major obstacle in tumor treatment. We describe a strategy for peptide-mediated delivery of compounds deep into the tumor parenchyma that uses a tumor-homing peptide, iRGD (CRGDK/RGPD/EC). Intravenously injected compounds coupled to iRGD bound to tumor vessels and spread into the extravascular tumor parenchyma, whereas conventional RGD peptides only delivered the cargo to the blood vessels. iRGD homes to tumors through a three-step process: the RGD motif mediates binding to alphav integrins on tumor endothelium and a proteolytic cleavage then exposes a binding motif for neuropilin-1, which mediates penetration into tissue and cells. Conjugation to iRGD significantly improved the sensitivity of tumor-imaging agents and enhanced the activity of an antitumor drug.

Targeting of albumin-embedded paclitaxel nanoparticles to tumors.

We have used tumor-homing peptides to target abraxane, a clinically approved paclitaxel-albumin nanoparticle, to tumors in mice. The targeting was accomplished with two peptides, CREKA and LyP-1 (CGNKRTRGC). Fluorescein (FAM)-labeled CREKA-abraxane, when injected intravenously into mice bearing MDA-MB-435 human cancer xenografts, accumulated in tumor blood vessels, forming aggregates that contained red blood cells and fibrin. FAM-LyP-1-abraxane co-localized with extravascular islands expressing its receptor, p32. Self-assembled mixed micelles carrying the homing peptide and the label on different subunits accumulated in the same areas of tumors as LyP-1-abraxane, showing that Lyp-1 can deliver intact nanoparticles into extravascular sites. Untargeted, FAM-abraxane was detected in the form of a faint meshwork in tumor interstitium. LyP-1-abraxane produced a statistically highly significant inhibition of tumor growth compared with untargeted abraxane. These results show that nanoparticles can be effectively targeted into extravascular tumor tissue and that targeting can enhance the activity of a therapeutic nanoparticle.

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.

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 compatibilities 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.

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.

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-beta-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.