The development of a wide spectrum of nano-scale technologies is beginning to change the foundations of disease diagnosis, treatment, and prevention. These technological innovations, referred to as nanomedicine have the potential to turn molecular discoveries arising from genomics and proteomics into widespread benefit for patients.
Nanomedicine is a large subject area and includes nanoparticles that act as biological mimetics (like functionalised carbon nanotubes), “nanomachines” (for example those made from interchangeable DNA parts and DNA scaffolds such as octahedron and stick cube), nanofibers and polymeric nanoconstructs as biomaterials and nano-scale microfabrication-based devices (like silicon microchips for drug release and micromachined hollow needles and two-dimensional needle arrays from single crystal silicon), sensors and laboratory diagnostics. Furthermore, there is a vast array of intriguing nano-scale particulate technologies capable of targeting different cells and extra-cellular elements in the body to deliver drugs, genetic materials, and diagnostic agents specifically to these locations.
For Imaging & Targeted Drug Delivery
Nanotechnology is an area of science devoted to the manipulation of atoms and molecules leading to the construction of structures in the nanometer scale size range (often 100 nm or smaller), which retain unique properties. Indeed, the physical and chemical properties of materials can significantly improve or radically change as their size is scaled down to small clusters of atoms. Small size means different arrangement and spacing for surface atoms, and these dominate the object’s physics and chemistry. Colloidal gold, ironoxide crystals, and quantum dots (QDs) semiconductor nanocrystals are examples of nanoparticles, whose size is generally in the region of 1–20 nm, and have diagnostic applications in biology and medicine.
Gold nanoparticles have application as quenchers in fluorescence resonance energy transfer measurement studies. For example, the distance-dependent optical property of gold nanoparticles has provided opportunities for evaluation of the binding of DNA-conjugated gold nanoparticles to a complementary RNA sequence. Iron oxide nanocrystals with super paramagnetic properties are used as contrast agents in magnetic resonance imaging (MRI), as they cause changes in the spin-spin relaxation times of neighboring water molecules, to monitor gene expression or detect pathologies such as cancer, brain inflammation, arthritis, or atherosclerotic plaques.
QDs can label biological systems for detection by optical or electrical means in vitro and to some extent in vivo. The fluorescence emission wavelength (from the UV to the near-IR) of QDs can be tuned by altering the particle size, thus these nanosystems have the potential to revolutionise cell, receptor, antigen, and enzyme imaging. Indeed, a recent report demonstrated the use of QDs for tracking metastatic tumor cell extravasation. Their large surface area-to-volume ratio offers potential for designing multifunctional nanosystems. Undoubtedly, application of such multi-wavelength optical nanotools may eventually aid our understanding of the complex regulatory and signaling networks that govern the behavior of cells in normal and disease states.
Nano as drug carriers
In addition, there are numerous engineered constructs, assemblies, architectures, and particulate systems, whose unifying feature is the nanometer scale size range (from a few to 250 nm). These include polymeric micelles, dendrimers, polymeric and ceramic nanoparticles, protein cage architectures, viral-derived capsid nanoparticles, polyplexes, and liposomes. First, therapeutic and diagnostic agents can be encapsulated, covalently attached, or adsorbed on to such nanocarriers. These approaches can easily overcome drug solubility issues, particularly with the view that large proportions of new drug candidates emerging from high-throughput drug screening initiatives are water insoluble.
Second, by virtue of their small size and by functionalising their surface with synthetic polymers and appropriate ligands, nanoparticulate carriers can be targeted to specific cells and locations within the body after intravenous and subcutaneous routes of injection. Such approaches, may enhance detection sensitivity in medical imaging, improve therapeutic effectiveness, and decrease side effects.
For Targeting
The network of blood and lymphatic vessels investing the body provides natural routes for the distribution of nutrients, clearing of unwanted materials, and delivery of therapeutic agents. Superficially, however, this network appears to provide little in the way of obvious controlled and specific access to tissues, and the science of these processes has been scant. Regardless of these limitations, nanoparticulate systems provide possibilities for access to cell populations and body compartments. When injected intravenously, particles are cleared rapidly from the circulation and predominantly by the liver and the spleen macrophages. This site-specific, but passive, mode of clearance is a facet of the immune cells’ primary scavenging role for particulate invaders and self-effete products. Opsonisation, which is surface deposition of blood opsonic factors such as fibronectin, immunoglobulins, and complement proteins, often aid particle recognition by these macrophages. However, size and surface characteristics of nanoparticles both play an important role in the blood opsonisation processes and clearance kinetics.
Small particle size also means large surface area. This may pose problems in terms of aggregation of primary nanoparticles in the biological environment, which subsequently determines the effective particle size and hence clearance kinetics. Indeed, dendrimers and QDs are well known to flocculate in biological media. Another case is interaction between certain lipid-based nanosystems and lipoproteins leading to dramatic size changes.
If confinement to the vascular system is necessary, then splenic filtration processes must be borne in mind. Splenic filtration at interendothelial cell slits is predominant. This is particularly true for rigid or nondeformable particles whose size exceeds the width of the cell slits (200–250 nm). Otherwise, opportunities are there for gaining efficient access to splenic red-pulp compartments with nanoparticles.
Recent developments in molecular biology have begun to reveal the wealth of information contained within blood and lymphatic vessels, and in particular that on the lumenal surface of endothelial cells. Molecular signatures related to particular vascular and lymphatic beds and types of endothelial cells have been identified, providing landmarks for circulating cells and molecules. These same signatures have now been exploited to direct therapeutic and diagnostic entities to selected pathological vessels, particularly those of cancer.
Macrophage as a target
The propensity of macrophages of the reticuloendothelial system for rapid recognition and clearance of particulate matter has provided a rational approach to macrophage-specific targeting with nanocarriers. The macrophage is a specialised host defense cell whose contribution to pathogenesis is well known. Alterations in macrophage clearance and immune effector functions contribute to common disorders such as atherosclerosis, autoimmunity, and major infections. The macrophage, therefore, is a valid pharmaceutical target and there are numerous opportunities for a focused macrophage-targeted approach. For example, although most microorganisms are killed by macrophages, many pathogenic organisms have developed means for resisting macrophage destruction following phagocytosis. In certain cases, the macrophage lysosome and/or cytoplasm is the obligate intracellular home of the microorganism, examples include Toxoplasma gondii, various species of Leishmania, Mycobacterium tuberculosis, and Listeria monocytogenes. Passive targeting of nanoparticulate vehicles with encapsulated antimicrobial agents to infected macrophages is therefore a logical strategy for effective microbial killing.
Macrophages and dendritic cells play critical roles in determining immunogenicity and the generation of appropriate immune responses. Systems, such as numerous polymeric and ceramic nanospheres, nanoemulsions, liposomes, protein cage architectures, and viral-derived nanoparticles act as powerful adjuvants if they are physically or covalently associated with protein antigens. After endocytic uptake of nanoparticles, macrophages partially degrade the entrapped antigens and channel peptides into the MHC molecules (class I or II) for processing and presentation. Thus, there is considerable potential for nanoparticulate adjuvants for the development of new-generation vaccines made either recombinantly or from synthetic peptide antigens that are less or nonimmunogenic in their own right. Genetic immunisation with nanoparticles has also received attention but the majority of attempts are based on cationic systems to allow DNA compaction.
Endothelium as a target
The concept of targeting to the blood vessels is an attractive one, particularly with the view that the endothelium plays an important role in a number of pathological processes including cancer (dysregulated angiogenesis), inflammation, oxidative stress and thrombosis. Indeed, a number of studies have demonstrated a level of control of arrest and distribution of passively targeted nanoparticles by specific endothelial cells, and these were linked to the surface properties of the carrier. For instance, early studies of polystyrene nanoparticles, designed to minimise liver cell uptake, indicated exclusive arrest by the bone marrow sinus lining endothelial cells in rabbits. Arrest was followed by receptor-mediated internalisation. Another example is the localisation of intravenously injected polysorbate 80-coated nanoparticles to murine and rat blood-brain endothelial cells. Recent studies have shown that cationic liposomes within 1 h of entering the circulation, are internalised into endosomes and lysosomes of endothelial cells in a characteristic organ- and vessel-specific manner. These patterns seem to bear no relationship to the morphological characteristics of the endothelium associated with a particular site, but probably reflect vessel-specific expression of receptors for which such particles, or their surface-associated blood proteins, are ligands.
Nevertheless, several studies have now combined the specificity of endothelial molecular markers with nanoparticles. For example, as a novel anti-angiogenic strategy targeted at solid tumors, some investigators have used a synthetic analog of αvß3 to target therapeutic genes complexed with cationic nanoparticles at tumor-associated endothelial cells. Similar approaches have now been extended for site-specific imaging with αvß3-targeted paramagnetic nanoparticles. This attempt detected and characterised early angiogenesis induced by minute solid tumors with magnetic resonance imaging. This is a valuable tool with which to phenotypic categorisation and patient selection as well as track the effectiveness of antitumor treatment regimens. Similarly, selective targeting of peptide-coated QDs to blood and lymphatic vessels in tumors have been demonstrated. Another interesting approach was the ability of NGR motif-decorated liposomes to specifically attack tumors by shutting down their blood supply.
Extravasation: targeting of solid cancers
The development of “stealth” technologies has provided opportunities for passive accumulation of intravenously injected nanoparticles (20–150 nm) in pathological sites expressing “leaky” vasculature by extravasation. Although, attempts have included delivery of drugs and imaging agents with different nanoscale technologies to the underlying parenchyma of injured arteries and rheumatoid arthritis, the majority of efforts are concentrated on solid tumors.
As a result of perfusion heterogeneity, the spatial distribution of stealth nanoparticles in solid tumors is heterogeneous and unpredictable. As has been elegantly demonstrated by Jain structural and functional abnormalities of blood and lymphatic vessels within solid tumors impede efficient delivery of not only systemic nanoparticles, but macromolecules. Already compromised by abnormal hydrostatic pressure gradients, compressive mechanical forces generated by tumor cell proliferation cause intratumoral vessels to compress and collapse. Tumor-specific cytotoxic therapy, reducing tumor cell number, may result in more efficient delivery, by decompressing these same vessels; however, this enhanced perfusion could provide a route for metastasis. Distribution, organisation and relative levels of collagen, decorin, and hyaluronan impede the diffusion of macromolecules and nanoparticles in tumors. Thus, diffusion of macromolecules and nanoparticles will vary with tumor types, anatomical locations, and possibly by factors that influence extracellular matrix composition and/or structure.
The issue of drug release from nanocarriers remains central to cancer chemotherapy. Therefore, application of a number of solid and polymeric nanoparticles for cancer drug delivery must be viewed cautiously since drug molecules may not be released from extravasated nanoparticles at sufficient rates. To date, the most effective approaches are described with liposomes and to some extent with polymeric micelles, although the latter constructs have a low encapsulation volume. There are a number of biochemical-based advances that can trigger drug release from accumulated liposomes at interstitial sites. One attractive approach is enzyme-mediated liposome destabilisation. For example, local concentration of secretory phospholipase A2 (sPLA2) is highly elevated in inflammatory and cancer tissues. The ester linkage in the sn-2 position of phospholipid is rapidly hydrolyzed by sPLA2.
This leads to production of a fatty acid and a lysolipid, which have a synergistic membrane perturbing and permeabilising effect. The activity of sPLA2 is much higher toward aggregated phospholipids (liposomes and micelles) than lipid monomers. sPLA2 can destabilise sterically protected liposomes bearing the substrate. So, by choosing an appropriate lipid composition one may control the extent of vesicle destabilisation by sPLA2. For example, stealth vesicles with entrapped cisplatin but composed of a nontoxic sPLA2 sensitive lipid prodrug (an ether lipid) were extremely effective in killing cancer cells in the presence of sPLA2, whereas the corresponding control formulation without sPLA2 sensitive lipids expressed insignificant cytotoxicity. Future efforts may consider combination strategies with sPLA2 sensitive and insensitive stealth liposomes, thus generating both rapid and prolonged interstitial drug release profiles in pathologies with elevated sPLA2 activity.
Nanoparticles for Drug Delivery
Breaching of the endosomal membrane is particularly important for priming MHC class I-restricted cytotoxic T lymphocyte responses, for survival of genetic materials against nuclease degradation in the lysosomal compartment, or for those drugs that must reach cytoplasm in sufficient quantities (as for treatment of cytoplasmic infections or reaching nuclear receptors) after endocytic delivery with nanoparticulate carriers. Here, there are advances in particle engineering too. For instance, nanoparticles made from poly(DL-lactide-co-glycolide) can escape the endo-lysosomal compartment within minutes of internalisation in intact form and reach the cytoplasm. The mechanism of rapid escape is by selective reversal of the surface charge of nanoparticles from the anionic to the cationic state in endo-lysosomes, thus resulting in a local particle-membrane interaction with subsequent cytoplasmic release. Another impressive approach for cytoplasmic delivery of nanoparticles is their surface manipulation with short peptides known as protein transduction domains such as HIV-1 TAT protein transduction domain (TAT PTD), which is a short basic region comprising residues 48–57, or heterologous recombinant TAT-fusion peptides. The electrostatic interaction between the cationic TAT PTD and negatively charged cell-surface constituents, such as heparan sulfate proteoglycans and glycoproteins containing sialic acids, is a necessary event before internalisation. After this ionic interaction, cellular uptake occurs by lipid raft-dependent macropinocytosis in a receptor-independent manner; this is followed by a pH drop and destabilisation of integrity of the macropinosome vesicle lipid bilayer, which ultimately results in the release of TAT-cargo into the cytosol. This mode of entry may further suggest the avidity of TAT PTD for glycophosphoinositol-anchored glycoproteins, which are present in lipid rafts, or binding to cholesterol membrane constituents that trigger macropinocytosis. An important feature of macropinosomes is that they do not fuse into lysosomes to degrade their contents. Although, these approaches have the potential to deliver and release drugs cytoplasmically for a sustained therapeutic effect in conditions such as cancer and stroke, possible cytotoxicity arising from the carrier components cannot be ruled out and warrants detailed investigation.
The Future
Nanotechnology is beginning to change the scale and methods of vascular imaging and drug delivery. Indeed, the NIH Roadmap’s ‘Nanomedicine Initiatives’ envisage that nano-scale technologies will begin yielding more medical benefits within the next 10 years. This includes the development of nano-scale laboratory-based diagnostic and drug discovery platform devices such as nano-scale cantilevers for chemical force microscopes, microchip devices, nanopore sequencing, etc.
The National Cancer Institute has related programs too, with the goal of producing nanometer scale multifunctional entities that can diagnose, deliver therapeutic agents, and monitor cancer treatment progress. These include design and engineering of targeted contrast agents that improve the resolution of cancer cells to the single cell level, and nanodevices capable of addressing the biological and evolutionary diversity of the multiple cancer cells that make up a tumor within an individual. Thus, for the full in vivo potential of nanotechnology in targeted imaging and drug delivery to be realised, nanocarriers have to get smarter. Pertinent to realising this promise is a clear understanding of both physicochemical and physiological processes. These form the basis of complex interactions inherent to the fingerprint of a nanovehicle and its microenvironment. Examples of which include carrier stability, extracellular and intracellular drug release rates in different pathologies, interaction with biological milieu, such as opsonisation, and other barriers en-route to the target site, be it anatomical, physiological, immunological or biochemical, and exploitation of opportunities offered by disease states.
Inherently, carrier design and targeting strategies may vary in relation to the type, developmental stage, and location of the disease. Toxicity issues are of particular concern but are often ignored. Therefore, it is essential that fundamental research be carried out to address these issues if successful efficient application of these technologies is going to be achieved. The future of nanomedicine will depend on rational design of nanotechnology materials and tools based around a detailed and thorough understanding of biological processes rather than forcing applications for some materials currently in vogue.
Nanopore sequencing
This is an ultra-rapid method of sequencing based on pore nanoengineering and assembly. A small electric potential draws a charged strand of DNA through a pore of 1–2 nm in diameter in an α–hemolysin protein complex, which is inserted into a lipid bilayer separating two conductive compartments. The current and time profile is recorded and these are translated into electronic signatures to identify each base. This method can sequence more than 1000 bases per second. This technology has much potential for the detection of single nucleotide polymorphisms, and for gene diagnosis of pathogens.
Cantilevers with functionalised tips
The enhanced spatial, force and chemical resolution of the atomic force microscope (AFM) and chemical force microscope can be taken into advantage for designing nanoscale diagnostic assays. The AFM probes intramolecular forces between a very fine and functionalised silicon or single-walled carbon nanotube tip, located at the end of a small cantilever beam, and a surface. The probe is attached to a piezoelectric scanner tube, which scans the probe across a selected area of the sample surface. Interamolecular and intraatomic forces between the tip and the sample cause the cantilever to deflect; cantilever deflection is then measured by a laser light reflected from the back of the cantilever to a detector. The tip can be chemically modified in order to probe a molecular structure of interest in drug discovery and measure biochemical interactions such as those between antigens and antibodies.
Microneedles
Micromachined needles and lancets with design adjustable bevel angles, wall thickness and channel dimensions have been engineered from single crystal silicon by combination of fusion bonding, photolithography, and ansiotropic plasma etching. This technology is being applied to painless drug infusion, cellular injection, and a number of diagnostic procedures (for example glucose monitoring).
Microchips for drug delivery
These are microfabricated devices that incorporate micrometer-scale pumps, valves, and flow channels and allow controlled release of single or multiple drugs on demand. These devices are particularly useful for long-term treatment of conditions requiring pulsatile drug release after implantation in a patient. The release mechanism is based on the electrochemical dissolution of thin anode membranes covering microreservoirs, which are filled with drugs. Thus, controlled delivery systems can be designed to release pulses of different drugs by using different materials for the membrane. Recently, microchip devices of 1.2 cm in diameter and thickness of approximately 500 µm with 36 drug reservoirs were fabricated from poly (L-lactic acid). The drug reservoirs were covered with poly (D, L-lactic-co-glycolic acid) membranes of different molecular masses.
Nucleic acid lattices and scaffolds
DNA can be programmed to self-assemble into an array of remarkable nanometer-scale structures different from the double helix. Stick cube, a construct shaped like a cube formed from sticks, and truncated DNA octahedron are two examples. For instance, the cube self-assembles from DNA fragments that are designed to adhere to one another. The free ends are connected by ligases, resulting in six closed loops, one for each face of the cube. Due to the helical nature of DNA, each of these loops is twisted around the loops that flank it, thus ensuring that the cube cannot come apart. Such scaffolds and assemblies can hold biological molecules in an ordered array for x-ray crystallography. This approach could be particularly useful for those materials that do not form a regular crystalline structure on their own (for example certain cell receptors that function as drug targets). These architectures could also hold molecule-size electronic devices, or be used to engineer materials with precise molecular configurations. Future efforts may lead to the design of DNA devices that can replicate, and DNA machines with moving parts as nanomechanical sensors, switches and tweezers.
Nanofibers as biomaterials
By applying molecular self-assembly, nanofibers of various structures and chemistries can be formed. Nanofibers may be designed to present high densities of bioactive molecules such as those which promote cell adhesion and growth. For example amphiphiles that present the pentapeptide epitope IKVAV, an amino acid sequence of laminin that promotes neurite adhesion, can self-assemble in aqueous media, or when injected directly into a tissue, to form fibers with a diameter of 5–10 nm. Indeed, these scaffolds were shown to induce rapid differentiation of cells to neurons, while discouraging the development of astrocytes. This presumably suggest that synthetic materials may have the ability to modulate selective gene expression.
Another interesting approach was the design of a synthetic collagen substitute, based on a material composed of a long hydrophobic alkyl group on one end and a hydrophilic peptide on the other that self-assembles into nanocylindrical structures. These nanocylinders guided the formation of hydroxyapatite crystallites with orientations and sizes similar to those in natural bone.
Carbon nanotubes
Carbon nanotubes belong to the family of fullerenes and consists of graphite sheets rolled up into a tubular form. These structures can be obtained either as single- (characterised by the presence of a single graphene sheet) or multi-walled (formed from several concentric graphene sheets) nanotubes. The diameter and the length of single-walled nanotubes may vary between 0.5–3.0 nm and 20–1000 nm, respectively. The corresponding dimensions for multi-walled nanotubes are 1.5–100 nm and 1–50 µm, respectively. Carbon nanotubes can be made water soluble by surface functionalisation. Molecular and ionic migration through carbon naotubes can occur, thus offering opportunities for fabrication of molecular sensors and electronic nucleic acid sequencing. Carbon nanotubes can apparently cross the cell membrane as ‘nanoneedles’ without perturbing or disrupting the membrane and localise into cytosol and mitochondria. However, the mechanisms are poorly understood.
A number of carbon nanotubes derivatives, such as tris-malonic acid derivative of the fullerence C60, express superoxide dismutase mimetic properties and are protective in cell culture and animal models of injury, including degeneration of dopaminergic neurons in Parkinson’s diseases and nervous system ischemia. The mechanism of action by C60 compounds appears to be through catalytic dismutation of superoxide. Furthermore, single-walled carbon nanotubes of 0.9–1.3 nm have been shown to block potassium channel subunits in a dose-dependent manner. However, not much is known with respect to in vivo toxicity of functionalised carbon nanotubes and their eventual intracellular fate. In the absence of detailed pharmacokinetic and toxicological studies, and their poor capacity to incorporate and release active compounds, the predicted benefits of carbon nanotubes in drug, antigen, and gene delivery remain hyped.
Quantum dots
These are nano-scale crystalline structures made from a variety of different compounds, such as cadmium selenide, that can transform the colour of light, and have been around since the 1980s. Quantum dots absorb white light and then re-emit it a couple of nanoseconds later at a specific wavelength. By varying the size and composition of quantum dots, the emission wavelength can be tuned from blue to near infrared. For example, 2nm quantum dots luminesce bright green, while 5nm quantum dots luminesce red. Quantum dots have greater flexibility, when compared to other fluorescent materials, and this makes them suitable for use in building nano-scale computing applications where light is used to process information. These structures offer new capabilities for multicolour optical coding in gene expression studies, high throughput screening, and in vivo imaging.
Dendrimers
These are highly branched macromolecules with controlled near monodisperse three-dimensional architecture emanating from a central core. Polymer growth starts from a central core molecule and growth occurs in an outward direction by a series of polymerisation reactions. Hence, precise control over size can be achieved by the extent of polymerisation, starting from a few nanometers. Cavities in the core structure and folding of the branches create cages and channels. The surface groups of dendrimers are amenable to modification and can be tailored for specific applications. Therapeutic and diagnostic agents are usually attached to surface groups on dendrimers by chemical modification.
Polymeric micelles
Micelles are formed in solution as aggregates in which the component molecules (e.g., amphiphilic AB-type or ABA-type block copolymers, where A and B are hydrophobic and hydrophilic components, respectively) are generally arranged in a spheroidal structure with hydrophobic cores shielded from the water by a mantle of hydrophilic groups. These dynamic systems, which are usually below 50 nm in diameter, are used for the systemic delivery of water-insoluble drugs. Drugs or contrast agents may be trapped physically within the hydrophobic cores or can be linked covalently to component molecules of the micelle.
Liposomes
These are closed vesicles that form on hydration of dry phospholipids above their transition temperature. Liposomes are classified into three basic types based on their size and number of bilayers. Multilamellar vesicles consist of several lipid bilayers separated from one another by aqueous spaces. These entities are heterogeneous in size, often ranging from a few hundreds to thousands of nanometers in diameter. On the other hand, both small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs) consist of a single bilayer surrounding the entrapped aqueous space. SUVs are less than 100 nm in size whereas LUVs have diameters larger than 100 nm. Drug molecules can be either entrapped in the aqueous space or intercalated into the lipid bilayer of liposomes, depending on the physicochemical characteristics of the drug. The liposome surface is amenable to modification with targeting ligands and polymers.
Nanospheres
These are spherical objects, ranging from tens to hundreds of nanometers in size, consisting of synthetic or natural polymers (collagen, albumin). The drug of interest is dissolved, entrapped, attached or encapsulated throughout or within the polymeric matrix. Depending on the method of preparation, the release characteristic of the incorporated drug can be controlled. As with liposomes, technology also allows precision surface modification of nanospheres with polymeric and biological materials for specific applications or targeting to the desired locations in the body.
Aquasomes (carbohydrate-ceramic nanoparticles)
These are spherical 60–300 nm particles used for drug and antigen delivery. The particle core is composed of nanocrystalline calcium phosphate or ceramic diamond, and is covered by a polyhydroxyl oligomeric film. Drugs and antigens are then adsorbed on to the surface of these particles.
Polyplexes/Lipopolyplexes
These are assemblies, which form spontaneously between nucleic acids and polycations or cationic liposomes (or polycations conjugated to targeting ligands or hydrophilic polymers), and are used in transfection protocols. The shape, size distribution, and transfection capability of these complexes depends on their composition and charge ratio of nucleic acid to that of cationic lipid/polymer. Examples of polycations that have been used in gene transfer/therapy protocols include poly-L-lysine, linear- and branched-poly(ethylenimine), poly(amidoamine), poly-ß-amino esters, and cationic cyclodextrin.
