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Previous studies from this group have shown that limit size lipid-based systems – defined as the smallest achievable aggregates compatible with the packing properties of their molecular constituents – can be efficiently produced using rapid microfluidic mixing technique. In this work, it is shown that similar procedures can be employed for the production of homogeneously sized unilamellar vesicular systems of 30–40 nm size range. These vesicles can be remotely loaded with the protonable drug doxorubicin and exhibit adequate drug retention propertiesin vitroandin vivo. In particular, it is demonstrated that whereas sub-40 nm lipid nanoparticle (LNP) systems consisting entirely of long-chain saturated phosphatidylcholines cannot be produced, the presence of such lipids may have a beneficial effect on the retention properties of limit size systems consisting of mixed lipid components. Specifically, a 33-nm diameter doxorubicin-loaded LNP system composed of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), 1,2-dipalmitoyl phosphatidylcholine (DPPC), cholesterol, and PEGylated lipid (DSPE-PEG2000) demonstrated adequate, stable drug retention in the circulation, with a half-life for drug release of ∼12 h. These results indicate that microfluidic mixing is the technique of choice for the production of bilayer LNP systems with sizes less than 50 nm that could lead to development of a novel class of ultra-small drug delivery vehicles. [ABSTRACT FROM PUBLISHER]

Efficient delivery of siRNA to cancer cells after systemic administration poses a significant challenge. While many methods of nucleic acid delivery have been described, encapsulation of siRNA in lipid nanoparticles (LNPs) represents the most clinically advanced delivery approach. Currently, there are two siRNA-LNP-based treatments (ALN-VSP and TKM-PLK1) in clinical trials targeting solid tumors, with additional studies ongoing for noncancer diseases. The consensus from these clinical studies is that siRNA-LNP represents safe and potent silencing systems. Improvements in LNP technology through development of more potent and biocompatible ionizable cationic lipids along with targeting lipids to mediate delivery of LNPs specifically to cancer cells are on the horizon. In combination with genomic screening, it is possible that within the next 5 years the pathogenic drivers of individual cancers will be identified and siRNA-based personalized medicines will be formulated to achieve successful treatment of cancer and other genetic diseases. [ABSTRACT FROM AUTHOR]

The encapsulation of conventional drugs in lipid nanoparticles (LNs) has been extensively utilized to enhance therapeutic activity by altering their pharmacokinetic (PK) and biodistribution (BD) properties. We have previously shown that the immunostimulatory activity of unmethylated cytidine-guanosine (CpG)-containing immunostimulatory oligodeoxynucleotides (ODN) is greatly enhanced when encapsulated in an LN (LN CpG-ODN). Here, we investigate the effect of circulation lifetime (determined by lipid composition) and drug-to-lipid (D/L) ratio of intravenously (i.v.) administered LN CpG-ODN on PK, BD, and cellular uptake and correlate these parameters with the immunostimulatory activity. Results from these studies show that despite significant differences in the circulation lifetime and the D/L ratio, the immune response is similar with respect to immune cell activation and cytolytic activity in the spleen and the blood compartments. Our findings indicate that the benefits of liposomal nanoparticles for the delivery of immunomodulatory drugs such as CpG-ODN are defined by a different paradigm than that for conventional drugs. [ABSTRACT FROM AUTHOR]

Recent studies have shown that the release properties of vincristine encapsulated in large unilamellar vesicles (LUV) can be regulated by varying the drug-to-lipid (D/L) ratio. In this work it is shown that the drug-to-lipid ratio technique for regulating drug release also applies to doxorubicin encapsulated in LUV. In particular it is shown that the half-times (T1/2) for doxorubicin release from distearoylphosphatidylcholine (DSPC)/cholesterol LUV in vitro can be increased more than six-fold by increasing the D/L ratio from 0.05 (wt/wt) to 0.39 (wt/wt). This behavior is consistent with the behavior expected for drugs that precipitate following accumulation into liposomes. It is shown that the release properties of ciprofloxacin—a drug that does not precipitate following accumulation into LUV—are not affected by the D/L ratio. It is also shown that the crystalline intravesicular doxorubicin precipitates observed as the D/L ratio is raised from 0.05 to 0.46 adopt increasingly unusual morphologies. Linear crystals are observed at lower D/L values, however triangular and rectangular variations are observed as the D/L ratio is increased, and induce considerable distortion in vesicle morphology. It is noted that trapping efficiency following uptake of external doxorubicin into LUV is reduced from nearly 100% at a D/L ratio of 0.05 (wt/wt) to less than 70% at an (initial) D/L ratio of 0.8 (wt/wt). It is suggested that this arises, at least in part, from membrane-disrupting effects of internal drug crystals as they increase in size. [ABSTRACT FROM AUTHOR]

Poly(ethylacrylic acid) (PEAA) is a pH-sensitive polymer that undergoes a transition from a hydrophilic to a hydrophobic form as the pH is lowered from neutral to acidic values. In this work we show that pH sensitive liposomes capable of intracellular delivery can be constructed by inserting a lipid derivative of PEAA into preformed large unilamellar vesicles (LUV) using a simple one step incubation procedure. The lipid derivatives of PEAA were synthesized by reacting a small proportion (3%) of the carboxylic groups of PEAA with C 10 alkylamines to produce C 10 -PEAA. Incubation of C 10 -PEAA with preformed LUV resulted in the association of up to 8% by weight of derivatized polymer with the LUV without inducing aggregation. The resulting C 10 -PEAA-LUV exhibited pH-dependent fusion and leakage of LUV contents on reduction of the external pH below pH 6.0 as demonstrated by lipid mixing and release of calcein encapsulated in the LUV. In addition, C 10 -PEAA-LUV exhibited pH dependent intracellular delivery properties following uptake into COS-7 cells with appreciable delivery to the cell cytoplasm as evidenced by the appearance of diffuse intracellular calcein fluorescence. It is demonstrated that the cytoplasmic delivery of calcein by C 10 -PEAA-LUV could be inhibited by agents (bafilomycin or chloroquine) that inhibit acidification of endosomal compartments, indicating that this intracellular delivery resulted from the pH-dependent destabilization of LUV and endosomal membranes by the PEAA component of the C 10 -PEAA-LUV. It is concluded that C 10 -PEAA-LUV represents a promising intracellular delivery system for in vitro and in vivo applications. [ABSTRACT FROM AUTHOR]

Cationic poly(ethylene glycol)-lipid conjugates(CPLs), a class of lipid designed to enhance the interaction of liposomes with cells, possess the following architectural features: 1) a hydrophobic lipid anchor of distearoylphosphatidylethanolamine(DSPE); 2) a hydrophilic spacer of poly(ethylene glycol); and 3) a cationic head group prepared with 0, 1, 3, or 7 lysine residues located at the distal end of the PEG chain, giving rise to CPL possessing 1, 2, 4, or 8 positive charges, respectively(CPL1 to CPL8). Previously we have described the synthesis of CPL, have characterized the postinsertion of CPL into PEG-containing LUVs and SPLP(stabilized plasmid-lipid particles), have shown significant increases in the binding of CPL-LUV to cells, and have observed dramatically enhanced transfection(up to a million-fold) of cells with CPL-SPLP in the presence of calcium[Chen et al.(2000) Bioconjugate Chem. 11, 433–437; Fenske et al.(2001) Biochim. Biophys. Acta 1512, 259–272; Palmer et al.(2003) Biochim. Biophys. Acta 1611, 204–216]. In the present study, we examine a variety of CPL properties(such as polarity and CMC) and characterize CPL-vesicular systems formed by extrusion and examine their interaction with cells. While CPL polarity was observed to increase dramatically with increasing charge number, CMC values were all found to be low, in the range of other PEGylated lipids, and exhibited only a small increase, going from CPL1(1.3µM) to CPL8(2µM). The CPLs were almost quantitatively incorporated into large unilamellar vesicles(LUVs) prepared by the extrusion method and were evenly distributed across the lipid bilayer. Lower levels of incorporation were obtained when CPLs were incubated with preformed liposomes(DSPC/Chol, 55:45) at 60°C. The binding of CPL-LUVs to BHK cells in vitro was found to be dependent on the distal charge density of the CPL rather than total surface charge. Liposomes possessing CPL4 or CPL8 were observed to bind efficiently to cell surfaces and enhance cellular uptake in BHK cells(as observed with both lipid and aqueous content markers), whereas those possessing CPL1 or CPL2 exhibited little or no binding. These results suggest new directions for the design of liposomal systems capable of in vivo delivery of both conventional and genetic(plasmid and antisense) drugs. [ABSTRACT FROM AUTHOR]

Currently available delivery systems for genetic drugs have limited utility for systemic applications. Cationic liposome/ plasmid DNA or oligonucleotide complexes are rapidly cleared from circulation, and the highest levels of activity are observed in `first pass' organs, such as the lungs, spleen and liver. Engineered viruses can generate an immune response, which compromises transfection resulting from subsequent injections and lack target specificity. A carrier, which can accumulate at sites of diseases such as infections, inflammations and tumours, has to be a small, neutral and highly serum-stable particle, which is not readily recognized by the fixed and free macrophages of the reticuloendothelial system (RES). This review summarizes lipid-based technologies for the delivery of nucleic acid-based drugs and introduces a new class of carrier systems, which solve, at least in part, the conflicting demands of circulation longevity and intracellular delivery. Plasmid DNA and oligonucleotides are entrapped into lipid particles that contain small amounts of a positively charged lipid and are stabilized by the presence of a polyethylene glycol (PEG) coating. These carriers protect nucleic acid-based drugs from degradation by nucleases, are on average 70 nm in diameter, achieve long circulation lifetimes and are capable of transfecting cells. [ABSTRACT FROM AUTHOR]