Dendrimers are branched, synthetic polymers with layered architectures that show promise in several biomedical applications. By regulating dendrimer synthesis, it is possible to precisely manipulate both their molecular weight and chemical composition, thereby allowing predictable tuning of their biocompatibility and pharmacokinetics. Advances in our understanding of the role of molecular weight and architecture on the in vivo behavior of dendrimers, together with recent progress in the design of biodegradable chemistries, has enabled the application of these branched polymers as anti-viral drugs, tissue repair scaffolds, targeted carriers of chemotherapeutics and optical oxygen sensors. Before such products can reach the market, however, the field must not only address the cost of manufacture and quality control of pharmaceutical-grade materials, but also assess the long-term human and environmental health consequences of dendrimer exposure in vivo.

Large unilamellar and oligolamellar vesicles are formed when an aqueous buffer is introduced into a mixture of phospholipid and organic solvent and the organic solvent is subsequently removed by evaporation under reduced pressure. These vesicles can be made from various lipids or mixtures of lipids and have aqueous volume to lipid ratios that are 30 times higher than sonicated preparations and 4 times higher than multilamellar vesicles. Most The use of phospholipid vesicles (liposomes) in biology and medicine, a promising new area of research (1), will depend to a large degree on technological improvements in the formation of vesicles of various sizes and properties. As more attempts are made to modify cellular physiology by introducing regulatory molecules into the cell or to improve chemotherapy in the whole animal, the need for a vesicle preparation that entraps a large percentage of the aqueous phase has become apparent.The original liposome preparations of Bangham et al. (2), consisting of multilamellar vesicles (MLV), have been admirably suited in defining many membrane properties (3, 4) and were the basis for the development of the sonicated unilamellar vesicles (SUV) (5). However, both preparations show a relatively low volume of entrapped aqueous space per mole of lipid and restricted ability to encapsulate large macromolecules. This is because in MLV most of the lipid is participating in the internal lamellae, and the close apposition of the adjacent concentric bilayers restricts the internal water space. In SUV, which are single-compartment vesicles, the ratio of surface area to encapsulated volume is so large that only a small aqueous volume per mole of lipid can be attained. Attempts to circumvent these shortcomings (6-8) have been only partially successful. The ethanol injection method produces vesicles of about the same size as SUV with the same shortcomings. The ether infusion technique produces large unilamellar vesicles with high captured volumes per mole of lipid, but the efficiency of encapsulation is relatively low. Other useful techniques for preparing large volume vesicles either use specialized conditions (9) or are restricted to a single phospholipid (10). Methods based upon solvent evaporation have been attempted in the past but have resulted in the formation of multilamellar vesicles (6,11,12). A method designed to form asymmetric vesicles by centrifugation of a suspension of dense aqueous inverted micelles through an organic solvent/water interface has been reported (13). The report indicated that the internal volume was small and the vesicles themselves relatively unstable (13). Techniques based upon the removal of detergents-yield vesicles slightly larger than SUV (14). These are suitable for membrane reconstitution experiments but, like the SUV, fail to encapsulate the aqueous phase efficiently. Recently a method that combines detergent dialysis and solvent evaporation has been described (15). This technique leaves an equal weight of detergent per phospholipid in the resu...

To understand how DNA is released from cationic liposome/DNA complexes in cells, we investigated which biomolecules mediate release of DNA from a complex with cationic liposomes. Release from monovalent[1,2-dioleoyl-3(1)-1(trimethylammonio)propane] or multivalent (dioctadecylamidoglycylspermine) lipids was quantified by an increase of ethidium bromide (EtBr) fluorescence. Plasmid sensitivity to DNAse I degradation was examined using changes in plasmid migration on agarose gel electrophoresis. Physical separation of the DNA from the cationic lipid was confirmed and quantified on sucrose density gradients. Anionic liposomes containing compositions that mimic the cytoplasmic-facing monolayer of the plasma membrane (e.g. phosphatidylserine) rapidly released DNA from the complex. Release occurred near a 1/1 charge ratio (-/+) and was unaffected by ionic strength or ion type. Water soluble molecules with a high negative linear charge density such as dextran sulfate or heparin also released DNA. However, ionic water soluble molecules such as ATP, tRNA, DNA, poly(glutamic acid), spermidine, spermine, or histone did not, even at 100-fold charge excess (-/+). On the basis of these results, we propose that after the cationic lipid/DNA complex is internalized into cells by endocytosis it destabilizes the endosomal membrane. Destabilization induces flip-flop of anionic lipids from the cytoplasmic-facing monolayer, which laterally diffuse into the complex and form a charge neutral ion pair with the cationic lipids. This results in displacement of the DNA from the cationic lipid and release of the DNA into cytoplasm. This mechanism accounts for a variety of observations on cationic lipid/DNA complex-cell interactions.

Cascade polymers also known as Starburst dendrimers are spheroidal polycations that can be synthesized with a well-defined diameter and a precise number of terminal amines per dendrimer. We show, using luciferase and beta-galactosidase containing plasmids, that dendrimers mediate high efficiency transfection of a variety of suspension and adherent cultured mammalian cells. Dendrimer-mediated transfection is a function both of the dendrimer/DNA ratio and the diameter of the dendrimer. Maximal transfection of luciferase are obtained using a diameter of 68 A and a dendrimer to DNA charge ratio of 6/1 (terminal amine to phosphate). Expression is unaffected by lysomotrophic agents such as chloroquine and only modestly affected (2-fold decrease) by the presence of 10% serum in the medium. Cell viability, as assessed by dye reduction assays, decreases by only 30% at 150 micrograms dendrimer/mL in the absence of DNA and about 75% in the presence of DNA. Under similar conditions polylysine causes a complete loss of viability. Gene expression decreased by 3 orders of magnitude when the charge ratio is reduced to 1:1. When GALA, a water soluble, membrane-destabilizing peptide, is covalently attached to the dendrimer via a disulfide linkage, transfection efficiency of the 1:1 complex is increased by 2-3 orders of magnitude. The high transfection efficiency of the dendrimers may not only be due to their diameter and shape but may also be caused by the pKa's (3.9 and 6.9) of the amines in the polymer. The low pKa's permit the dendrimer to buffer the pH change in the endosomal compartment. The characteristics of precise control of structure, favorable pKa's, and low toxicity make the dendrimers suitable for gene-transfer vehicles.

Four cationic polymers used to deliver DNA into cultured dendrimer generally appeared as clusters in electron cells: polylysine, intact polyamidoamine dendrimer, fracmicrographs; their diameters in solution were larger than tured polyamidoamine dendrimer and polyethylenimine, 1000 nm, which suggests that their toroidal complexes are examined for their ability to interact with DNA. Comaggregate in solution. The cationic polymers bind to DNA plexes between the polymers and DNA were examined in a stoichiometry that is nearly 1:1 in primary amines to using electron microscopy. Similar toroidal structures with DNA phosphates. The apparent binding of all cationic polydiameters of 55 ± 12 nm were formed from all of the catmers to DNA decreases linearly with increasing ionic ionic polymers with DNA. The DNA complexes of the fracstrength, up to 0.8 M NaCl. Thus, at the concentrations tured dendrimer and polyethylenimine were observed as studied, these polymers interact electrostatically with DNA single, distinct units; their apparent diameters in solution forming a unit structure with toroidal morphology; the as measured by dynamic light scattering ranged from 90 extent of aggregation of the unit structures in solution to 130 nm. The DNA complexes of polylysine and intact depends upon the characteristics of the individual polymer.

Transfection of cultured cells has been reported using complexes between DNA and spherical cationic polyamidoamine polymers (Starburst dendrimers) that consist of primary amines on the surface and tertiary amines in the interior. The transfection activity of the dendrimers is dramatically enhanced (> 50-fold) by heat treatment in a variety of solvolytic solvents, e.g., water or butanol. Such treatment induces significant degradation of the dendrimer at the amide linkage, resulting in a heterodisperse population of compounds with molecular weights ranging from the very low (< 1500 Da) to several tens of kilodaltons. The compound facilitating transfection is the high molecular weight component of the degraded product and is denoted as a "fractured" dendrimer. Transfection activity is related both to the initial size of the dendrimer and its degree of degradation. Fractured dendrimers exhibit an increased apparent volume change as measured by an increase in the reduced viscosity upon protonation of the terminal amines as pH is reduced from 10.5 to 7.2 whereas intact dendrimers do not. Dendrimers with defective branching have been synthesized and also have improved transfection activity compared to that of the intact dendrimers. For a series of heat-treated dendrimers we observe a correlation between transfection activity and the degree of flexibility, computed with a random cleavage simulation of the degradation process. We suggest that the increased transfection after the heating process is principally due to the increase in flexibility that enables the fractured dendrimer to be compact when complexed with DNA and swell when released from DNA.

CONSPECTUS Chemotherapy can destroy tumors and arrest cancer progress. Unfortunately, severe side effects—treatment is usually a series of injections of highly toxic drugs—often restrict the frequency and size of dosages, much to the detriment of tumor inhibition. Most chemotherapeutic drugs have pharmacokinetic profiles with tremendous potential for improvement. Water-soluble polymers offer the potential to increase drug circulation time, improve drug solubility, prolong drug residence time in a tumor, and reduce toxicity. Cytotoxic drugs that are covalently attached to water-soluble polymers via reversible linkages more effectively target tumor tissue than the drugs alone. Macromolecules passively target solid tumor tissue through a combination of reduced renal clearance and exploitation of the enhanced permeation and retention (EPR) effect, which prevails for fast-growing tumors. Effective drug delivery involves a balance between (i) elimination of the polymeric drug conjugate from the bloodstream by the kidneys, liver, and other organs and (ii) movement of the drug out of the blood vasculature and into the tumor (that is, extravasation). Polymers are eliminated in the kidney by filtration through pores with a size comparable to the hydrodynamic diameter of the polymer; in contrast, the openings in the blood vessel structures that traverse tumors are an order of magnitude greater than the diameter of the polymer. Thus, features that may broadly be grouped as the “molecular architecture” of the polymer—such as its hydrodynamic volume (or molecular weight), molecular conformation, chain flexibility, branching, and location of the attached drug—can greatly impact elimination of the polymer from the body through the kidney but have a much smaller effect on the extravasation of the polymer into the tumor. Molecular architecture can in theory be adjusted to assert essentially independent control over elimination and extravasation. Understanding how molecular architecture affects passage of a polymer through a pore is therefore essential for designing polymer drug carriers that are effective in passively delivering a drug payload while conforming to the requirement that the polymers must eventually be eliminated from the body. In this Account, we discuss examples from in vivo studies that demonstrate how polymer architectural features impact the renal filtration of a polymer as well as tumor penetration and tumor accumulation. In brief, features that inhibit passage of a polymer through a pore—such as higher molecular weight, decreased flexibility, and an increased number of polymer chain ends—help prevent elimination of the polymer by the kidneys and can improve blood circulation times and tumor accumulation, thus improving therapeutic effectiveness.

The antitumor effect of doxorubicin (DOX) conjugated to a biodegradable dendrimer was evaluated in mice bearing C-26 colon carcinomas. An asymmetric biodegradable polyester dendrimer containing 8 -10 wt % DOX was prepared. The design of the dendrimer carrier optimized blood circulation time through size and molecular architecture, drug loading through multiple attachment sites, solubility through PEGylation, and drug release through the use of pH-sensitive hydrazone linkages. In culture, dendrimer-DOX was >10 times less toxic than free DOX toward C-26 colon carcinoma cells after exposure for 72 h. antitumor ͉ molecular architecture ͉ therapeutic effect ͉ nanomedicine ͉ dendrimer prodrug