Nanotechnology in Biomedicine.doc

Nanotechnology in BiomedicineJohn Hoffman, Melvin Zin, and Yi HeSynopsisBiodegradable polymers have been have been used as drug delivery devices for over a decade. Due to their small size and physical interaction with surrounding solvent, these structures have shown the ability to avoid the reticuloendothelial system and, in the case of tumor targeting, utilize the enhanced permeability and retention (EPR) effect. Being organic molecules, functional groups are available for attaching moieties designed to enhance target specificity.In the last 10 years, the field of diagnostics has been impacted greatly by the use of nanomaterials in assays for gases, metal ions, and DNA and protein markers for various diseases. The need to overcome the limitations of conventional techniques for diagnostics in terms of sensitive and selective detection has driven the intense research on the applications of metallic and magnetic nanoparticles and semiconductor quantum dots as both passive (probes, fluorescent markers) and active (in assays involving energy transfer) components in medical devices.The application of nanotechnology offers great potential advantages in drug discovery and development. It can significantly enhance the costly drug screening process by providing a stable and effective imaging carrier. They could be attached to readily to a drug molecule and enable the tracking of the molecule in a cell or in other biological samples. Also, it can be conjugated with some drug receptors of biological interest to give a highly sensitive imaging for studying the behavior of these receptors, which is crucial to targeting new drug candidates. In this paper we will review the applications of nanotechnology in drug delivery, biodiagnostic, and bioimaging.1 Drug DeliveryChemotherapy has arisen as the primary treatment for malignant disease and has shown promise in the delivery of small molecule, protein and nucleic acid based drugs. Several diseases are susceptible to systemic chemotherapy, but require multiple doses to achieve remission. These chemotherapeutic agents show dose-response behavior (where tissue response is proportional to the local concentration of drug) and remission requires an effective dose be delivered to the target tissue. Unfortunately, healthy tissue is also susceptible to these systemic treatments in a dose-response manner, but the response of normal tissue is usually offset to higher concentrations of the drug. The difference in dose required for a response in diseased versus healthy tissue is defined as the therapeutic index and, in several chemotherapies, this index is very small. The selectivity of the drug to specific tissue types defines the index and typically proliferative tissues, such as bone marrow and gastrointestinal mucosa, are the most susceptible. The selectivity is dependent on several factors, with cytokinetics, tissue susceptibility to programmed cell death and unique metabolic pathways in diseased tissues contributing the most drug selectivity. With a predominant lack of selectivity in chemotherapy, chronic chemotherapy results in severe systemic toxicity. With the goal of improving selectivity, as well as stabilizing formulations for the in vivo aqueous environment and protection of drugs from physiological degradation, several approaches have been taken to drug delivery. Amphiphilic molecules and polymers have been used as carriers, but biocompatible amphiphilic polymers are most commonly used because current polymer construction techniques offer sublime tailoring of chemical activity and achieve optimal medicinal effects. The chemical composition and architecture can be tailored to accommodate drugs with varying hydrophobicity, molecular weight, and pI (to name a few) as well as provide conjugation sites for crosslinking and attachment of targeting moieties. Furthermore, polymers can be tailored for specific biodegradation and controlled drug release kinetics.Over the last two decades, biodegrabable polymeric nanoparticles have emerged as candidates for selective drug delivery. Nanoparticles offer a wide range of drug loading options, where the drug can be dissolved, encapsulated or covalently attached within the nanoparticle matrix leading to a wide range of particle morphologies with tailored release properties. Also, their coronas are easily modified to achieve increased biocompatibility and stability in vivo, while delivering unique biodistribution through the incorporation of targeting moieties. Lastly, as the particle degrades, monomer units are non-toxic and either easily cleared from the body by the renal system, or incorporated into metabolic pathways. In this paper we will discuss the polymers used to create biodegradable drug delivery vectors and the techniques for creating nanoparticles. We will discuss the various loading options leading to controlled release. We will investigate various coronal modifications presented in the literature that lead to targeted delivery and biodistribution and lastly, the ability of these systems to be cleared from the body after delivery. 1.1 Biodegradable Polymers in Drug DeliveryFor the purposes of this paper, biodegradable polymers are ones whose degradation in biological environments is achieved through hydrolysis. While other modes of degradation are available, such as thermal oxidation or photolysis, these modes are not typically found in the human body (or other animal). The use of polymers in vivo is limited, as several requirements are imposed on the polymer through federal regulation, such as biocompatibility, non-toxicity, sterilizability, and effectiveness. The polymers discussed in this paper are unique in that their degradation products have been found to be non-toxic, have excellent biocompatibility and have been shown to be effective in drug delivery.Biodegradable polymers can be either natural or synthetic, were synthetic polymers offer a wider range of options as their synthesis is tailorable. The polylactides Poly(glycolic acid) (PGA) and poly(lactic acid) (PLA), along with the co-polymer Poly(lactide-co-glycolide) (PLGA), are the most commonly used biodegradable synthetic polymers as they both have extensive clinical applications and have shown to be effective in implantation drug delivery1. The degradation rate of these polymers depends on several factors. Most important are the relative hyrdophobicities, polymer crystalinity, and pH of the aqueous solvent2. The degradation products of PLA, PGA and PLGA are lactic acid and glycolic acid, which are biologically compatible and metabolizable moieties that are eventually removed from the body by the citric acid cycle3. Polymer biodegradation products are formed at a very slow rate, and hence they do not affect the normal cell function4.Poly(e-caprolactone) (PCL) is another aliphatic polyester, whose repeating unit is substantially larger than either PLA or PGA. PCL has found several uses as in sutures and drug delivery. PCL was first used by Pitt et al5, 6 in the late 1970s for the controlled release of steroids and narcotic antagonists. PCL is advantageous as a drug delivery vehicle for several reasons. Lemoine et al7 show PCL has great persistence under various storage conditions, much more so than PLA, PGA and PLGA. Also, as we will discuss in more detail later, hydrophobic nanoparticles have an increased capacity for drug loading, but with relatively slow drug release (which is not always a limitation, depending on the application) and issues with solubility. Lastly, although not biodegradable, poly(ethylene glycol) is widely used as a hydrophilic block or surface agent and is one of the few polymers approved by the US FDA as a drug delivery agent. Fred Davis8 began conjugating low molecular weight PEGs to proteins in the late 1970s while Ed Merrill9 was incorporating PEG into hydrogels to improve biocompatibility in the early 1980s. There is ample literature on the subject of grafting PEG to nanoparticles to increase solubility and biodistribuition, while decreasing protein adsorption and immune response in vivo. For example, Li et al10 show PEG-PLGA nanoparticles as peptide drug carriers where the incorporation of PEG not only increases the stability of the proteins within the particle during and after preparation and increases the drug release kinetics, but also changes the biodistribution of the particles in a rat model. 1.2 Nanoparticle Formulation and Drug LoadingSeveral techniques have been used to formulate nanoparticles. Techniques usually rely on forming particles from pre-polymerized units, or in situ polymerization of monomers contained within surfactant micelles. Emulsions have shown to be the most widely used technique where preformed polymer is added to solvent, typically dichloromethane, chloroform or ethyl acetate. The drug is then loaded into the polymer solution. This mixture is added to an aqueous solution with high surfactant concentration generating micelles. The solution in then heated, with either continuous mixing or applied pressure and the solvent is evaporated leaving surfactant coated nanoparticles with loaded drug. This protocol leads to nanoparticles with diameter less that 500 nm. To create particles with smaller diameters, water-soluble solvents are added to the original polymer-solvent solution and, when introduced to the aqueous environment, create turbulence at the oil-water interface that leads to decreased particle sizes with increasing concentration. Other techniques include lowering the polymer concentration and increasing the surfactant concentration or lowering the polymer molecular weight11. Peptide drugs and genetic material (DNA and various forms of RNA) have great potential as therapies if they can be delivered to diseased tissues intracellularly12. Their use is limited as these molecules are highly susceptible to proteolytic enzymes in the GI tract, their biological half life is short and their ability to transport across biological membranes is poor. Nanoparticles are thought to be suitable carries for these molecules, but several hurdles exist. First, the widely used emulsion techniques for particle formation are problematic as these biomolecules tend to denature at the aqueous/organic interface. Next, peptides and proteins, which are usually have hydrophilic exteriors, are not easily loaded into the hydrophobic interiors of the nanoparticles and tend to denature once loaded. Lastly, the pH within the interior of a degrading nanoparticle can dip as low as pH 3, which can affect protein stability. This issue can be overcome by protein complexation with zinc and control of PLGA microclimate pH with antacid excipients13.The most commonly used method to overcome the solvent incompatibility issue is the water-in-oil-in-water emulsion technique14, 15. Here, the hydrophilic drug is dissolved into an aqueous phase, which is then emulsified in organic solvent, typically ethyl acetate or methylene chloride. This phase is then emulsified in an aqueous phase containing a stabilizer, such as Pluronic F-68/DDW. Several other emulsion techniques have been described, such as the solid-in-oil-in water16 and solid-in-oil-in-oil17, as well as other alternatives, such as spray freeze drying18, atomization of a drug/polymer organic solution in a high pressure carbon dioxide (CO2) environment19 and nanoprecipitation20.All of these techniques have shown effective in creating particles with diameters ranging from 85 to 500 nanometers, drug loading and due to the degradation profile of the nanoparticle, lead to controlled release of drugs both in vitro and in vivo. 1.3 Targeting and Release1.3.1 Controlled ReleaseThe release profile from nanoparticles depends mostly on the method of drug incorporation. We have focused primarily on particles with uniform distribution of drug and these matrix style devices show release dependent on both diffusion and matrix biodegradation. Peppas21 proposed the following equation for drug release kinetics:Where Mt/M0 is the fraction of drug released up to time t, K is a constant incorporating the structural and geometric characteristics of the release device, and n is the release exponent indicative of the mechanism of release and ranges from 0.5 to 1.0 (zero-order).This relationship has proven useful for describing the release of small molecules and macromolecules. In general, the smaller the particle size, the faster the release as small particle size leads to higher surface to volume ratio, increasing degradation and diffusion. Release also depends on the hydration interactions between the drug and the polymer where attractive hydration forces (hydrophobic and hydrophobic) tend to decrease release rates. 1.3.2 Avoiding Host Immune ResponseWithin the human body, foreign substances are labeled by the host immune system through the binding of complement proteins and antibodies to the surface and cleared by macrophages in the organs comprising the mononuclear phagocytes system (MPS) (process known as opsonization). The three main organs comprising the MPS are the lungs, spleen and liver. If the target tissue/organ is not within this system, the addition of a densely packed hydrophilic polymer, such as PEG, has been shown to significantly reduce protein adsorption to the surface and subsequent uptake by macrophages10, 19, 22-24. 1.3.3 The Enhanced Permeation and Retention (EPR) EffectTumors are shown to have defective vascular architecture and poor lymphatic drainage. The defective vasculature arises from the increased nutrient requirements for fast growing tumor tissues and results in improved transport of molecules through the epithelial layer. Nanoparticles with hydrophilic coatings have exploited this hyperpermeability and been shown to partition within tumors25-27. The reduction in lymphatic drainage prevents the clearance of nanoparticles from the tumor tissues and ensured their persistence. The enhanced metabolic rate in cancer tissues also leads to an accumulation of lactic acid within the tumor, thus decreasing pH and increasing the degradation rate of the polylactide nanoparticles.1.3.4 Targeting Disease Specific Cell Surface ReceptorsWhile initial work with biodegradable nanoparticles relied on a local chemotherapeutic effect, direct targeting of tissues was not available28. Diseased cells, especially tumors, have a larger than normal need for vitamin and nutrients and therefore express a wide variety of receptors on their surface than can be used for targeting. Once the targeting moiety interacts with the cell surface receptor, receptor mediated endocytosis allows for the uptake of the nanoparticle and facilitates intracellular release of drug cargo29-32. The