Nanoparticles for Convection-Enhanced Drug Delivery into the Human Brain

1. Nanoparticles for Convection-Enhanced Drug Delivery into the Human Brain Prepared for the Laboratory for Product and Process Design Created by Eric Lueshen Presented on June 17, 2009

2. Overview of Presentation • Background information. • Convection-Enhanced Delivery (CED) • Types of Nanoparticles • Nanoparticle (NP) use in CED and its benefits • Important NP physicochemical properties and their influence on brain diffusivity and CED. • Preparation methods of drug-loaded NPs and their comparisons. • Modification of nanoparticles. • Three different preparative methods of surface-modified NPs. • Nanoparticle characterization. • Future research goals.

3. Convection-Enhanced Delivery • Utilizes an applied external pressure gradient to induce fluid convection in the brain. • Therapeutic fluid is administered via a small catheter using a pump. • Results in larger volume of distribution compared to classic diffusion alone, and a higher infusate concentration over a longer distance. Fig 1. Schematic representation of CED into a rat. [4]

4. Types of Nanoparticles [4]

5. Nanoparticle use in CED • Allows for the effective delivery of therapeutic drugs into the brain while bypassing the Blood Brain Barrier (BBB). • NPs can be modified to perform specific tasks: • Target certain cells or other areas of interest • Immunotherapy • Gene therapy • Imaging techniques to document drug distribution • Immense possibilities for developing new treatment methods to combat cancer, infections, metabolic and autoimmune diseases, and central nervous system (CNS) diseases.

6. Important NP Properties to Consider for CED of Drugs into the Brain ECM: extracellular matrix; PEG: polyethylene glycol; bFGF: basic fibroblast growth factor; LNC: lipid nanocapsules [4]

7. Drug-Loaded NP Preparation Methods • Two main preparation method categories each containing different techniques to prepare NPs: • Formulation requiring a polymerization reaction • Emulsion polymerization • Interfacial polymerization • Prepared directly from a macromolecule or preformed polymer (either synthetic or natural): • Synthetic • Solvent displacement and interfacial deposition • Salting out • Emulsion/solvent diffusion • Natural • Chitosan [5]

8. Emulsion Polymerization • Monomers and drugs are dissolved in an aqueous continuous phase. • Initiation occurs when dissolved monomer molecules collide with an initiator molecule (ion or free radical). • Alternatively, monomer molecules can be transformed into initiating radicals by high-energy radiation. • The resulting colloidal suspension is then concentrated by evaporation under vacuum. • Drug-encapsulated NPs formed from this method: • Poly(methylmethacrylate) [PMMA] copolymers with Doxorubicin (cancer chemotherapy drug) • Poly(isobutylcyanoacrylate) [PIBCA] with Ampicillin (used to treat bacterial infections) [5]

9. Interfacial Polymerization • Monomers and drugs are dissolved in an emulsion mixture. • This mixture is then extruded into a well-stirred solution with or without the presence of a surfactant. • Nanocapsules are formed spontaneously by polymerization or monomers after contact with initiating ions present in the mixture. • The resulting colloidal suspension is then concentrated by evaporation under vacuum. • High efficiency of drug encapsulation. • Drug-encapsulated NPs formed from this method: • Poly(isohexylcyanoacrylate) [PIHCA] with Phthalocyanines (photoactivatable cytotoxic compounds used in photodynamic tumor therapy) [5]

10. Solvent Displacement & Interfacial Deposition • Involves the precipitation of a preformed polymer from an organic solution and the diffusion of the organic solvent in the aqueous medium (with/without surfactant). • High encapsulation efficiencies; simplicity. • Drug-encapsulated NPs formed from this method: • Poly(lactic acid)-poly(glycolic acid) [PLGA] copolymer with Paclitaxel or Doxorubicin (both are cancer chemotherapy drugs), etc. Fig 3. Schematic representation of the solvent displacement technique. **Surfactant is optional. ***In interfacial deposition method, a fifth compound was introduced only on preparation of nanocapsules. [5] [5]

11. Salting Out with Synthetic Polymers • Polymer and drug are dissolved in a solvent, which is emulsified into an aqueous gel containing the salting-out agent (electrolytes such as CaCl2 or non-electrolytes such as sucrose) and a colloidal stabilizer such as polyvinylpyrrolidone (PVP). • Emulsion is diluted with aqueous solution to enhance the diffusion, thus inducing the formation of nanospheres. • Solvent and salting-out agent are then eliminated by cross-flow filtration. • Salting out minimizes stress to protein encapsulants and does not require an increase of temperature. • Drug-encapsulated NPs formed from this method: • Poly(lactic acid) [PLA] with Savoxepin (antipsychotic drug) Fig 4. Schematic representation of the salting-out technique. [5] [5]

12. Emulsion/Solvent Diffusion (ESD) • Polymer is dissolved in a partially water-soluble solvent (such as propylene carbonate) and saturated with water to ensure initial thermodynamic equilibrium of both liquids. • The polymer-water saturated solvent phase is emulsified in an aqueous solution containing stabilizer, leading to solvent diffusion to the external phase and the formation of NPs. • Solvent is eliminated by evaporation or filtration. • Drug-encapsulated NPs formed from this method: • Poly(lactic acid)-poly(glycolic acid) [PLGA] copolymer with Doxorubicin (cancer chemotherapy drugs) or p-THPP (coenzyme). Fig 5. Schematic representation of the ESD technique. [5] [5]

13. Chitosan Nanoparticles • Produced by a promoting gelation in an emulsification-based preparation method. • Chitosan NPs have been developed to encapsulate proteins used for anticancer agents, vaccines, etc. Fig 7. Schematic representation of chitosan NPs preparation by the emulsification technique. [5] [5]

14. Advantages & Drawbacks of the Different Preparation Methods Table 1. General advantages and drawbacks of the preparation methods. [5] [5]

15. Modification of Nanoparticles • NP coatings should be hydrophilic, biocompatible, non-toxic, non-detective by immune systems, and induce few side effects. • Generally, NPs are coated with a synthetic polymer. • Examples of synthetic polymers: • poly(cyanoacrylate) [PCA] • poly(lactic acid) [PLA] • poly(lactide-co-glycolic acid) [PLGA] • Significantly increase the distribution of NPs due to protection from the host immune system, etc. by reducing NP interactions with proteins. • NPs can also be conjugated with functional ligands or proteins for site-specific delivery. • After coating NPs, purify the NP suspensions by centrifugation or by ultrafiltration and then freeze-dry the NPs for storage. (consider using a lyoprotectant)

16. Three Preparative Methods of Surface-Modified Nanoparticles • Method I: Surface modification and particle formation occur simultaneously by the emulsification of an oil phase with polymer in an aqueous phase with amphiphilic materials (poloxamer, polysorbate, poly(ethylene glycol) [PEG], polysaccharides, etc.). These amphiphilic materials adsorb on the surfaces and form a hydrophilic layer. • Very simple and basic method for the functionalization of NPs. Fig 8. Schematic representation of the first method for preparation of surface-modified NPs. [6] [7]

17. Examples of Nanoparticles Prepared by Method I [7]

18. Three Preparative Methods of Surface-Modified Nanoparticles continued… • Method II: Surface of NP is modified with coupling agents after particle preparation. The polymer used should have at least one reactive group located on the NP’s surface for efficient conjugation. The coupling agent is dissolved in the continuous phase of a NP suspension and it then activates the reactive groups of NPs. These activated groups are then conjugated with functional materials like drugs, ligands, enzymes, and so on. Fig 9. Schematic representation of the second method for preparation of surface-modified NPs. [6] [7]

19. Three Preparative Methods of Surface-Modified Nanoparticles continued… • Method III: The polymers conjugated with hydrophilic segments form core-shell structured NPs and show a long circulating ability. The hydrophobic core gives a domain for the entrapment of hydrophobic drugs. Various types of tri/di-block copolymers have been synthesized. • Good way to make relatively small NPs. • Offers an easy way to control the drug release rate. Fig 10. Schematic representation of the third method for preparation of surface-modified NPs. [6] [7]

20. Conjugation Strategies of a Functional Moiety to Polymer via Methods II and III • In the cases of Methods II and III, a chemical reaction between the polymer and functional moiety is involved. • Involves the reaction between carboxyl groups and amine groups to form amide linkages. carboxyl group general amine structure amide [7]

21. Fig 11. Several chemical strategies using coupling agent for conjugation. [6] [7]

22. Nanoparticle Characterization • The physicochemical properties of NPs affect their functional efficiency and therefore need to be well characterized. • Important parts of NP characterization: • Surface charge (zeta potential) • Hydrophobicity • Size • Morphology • Stability • Composition • Amount of drug and targeting peptides • Imaging [6]

23. Zeta Potential & Hydrophobicity • Zeta potential (determines colloidal system’s stability) • The surface charge of electrostatically-stabilized NP tends to be decreased as the modification degree increases. • If the value is large = stable system • If the value is small = system will agglomerate • X-ray Photoelectron Spectroscopy (XPS) can be used to help calculate NP zeta potentials and hydrophobicities. • XPS measures the elemental composition, empirical formula, chemical state and electronic state of the elements that exist within a nanoparticle. • Spectra obtained by irradiating NP with a beam of X-rays while simultaneously measuring the kinetic energy and number of electrons that escape from the top 1-10 nm of the NP. [8]

24. X-ray Photoelectron Spectroscopy Fig 11. Basic components of XPS system. [http://en.wikipedia.org/wiki/X-ray_photoelectron_spectroscopy]

25. Size, Morphology and Surface Appearance • Scanning electron microscopy (SEM). • Images the sample surface by scanning it with a high-energy beam of electrons in a rectangular scan pattern. • The electrons interact with the atoms and produce signals that contain information about the NP surface topography, composition, size, etc. Fig 12. SEM of pollen grains. [http://en.wikipedia.org/wiki/Scanning_electron_microscope]

26. Size, Morphology and Surface Appearance continued… • Transmission electron microscopy (TEM). • Electron beam transmitted through an ultra thin NP specimen, interacting with the NP as it passes through. • Image is formed from these interactions and is magnified & focused onto an imaging device. • TEMs are capable of imaging at a significantly higher resolution than light microscopes. Fig 13. Basic TEM optical components. [http://en.wikipedia.org/wiki/Transmission_electron_microscopy]

27. Morphology, Stability & Composition • Differential scanning calorimetry (DSC): • The difference in amount of heat required to increase the temperature of a sample and reference are measured as a function of temperature. • Both sample and reference are maintained at nearly the same temperature. • Used to study crystallization events, polymer cross-linking, polymer degradation and composition, oxidation (stability and optimum storage conditions), etc. Fig 14. Differential Scanning Calorimeter. [http://en.wikipedia.org/wiki/Differential_scanning_calorimetry]

28. Analysis of Drug and Targeting Peptide Release • Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) • Used to separate proteins according to their electrophoretic mobility. • Proteins denatured and given a negative charge by SDS (an anionic detergent). • Denatured proteins are applied to one end of a layer of polyacrylamide gel submerged in a buffer. • Electric current is applied across the gel, causing the negatively-charged proteins to migrate across the gel towards the anode. Depending on their size, each protein will move differently through the gel matrix. Fig 15. Picture of an SDS-PAGE. [http://en.wikipedia.org/wiki/SDS-PAGE]

29. Drug Loading Capacity (LC) & Drug Loading Efficacy (LE) • Nanoparticle suspension is centrifuged and then the protein in the supernatant is quantified by a protein assay. [8]

30. Imaging with MRI • NPs can be labeled by incorporating a marker in the NP membrane and/or loaded by encapsulation during NP preparation methods. • Radiolabeling can give information about brain residence time and in situ degradation. • MRI contrast agents (such as iron oxides) as markers allow real-time imaging. • Magnetic Resonance Imaging (MRI): • Once inside the magnetic field of the scanner, the body’s water protons align with the direction of the field. • A second electromagnetic field is briefly turned on causing the protons to absorb some energy. When this field is turned off the protons release this energy which is detected by the scanner. • Contrast agents enhance the appearance of blood vessels, tumors and inflammation.

31. Future Research Goals • Continue to learn about the numerous methods and compounds used in convection-enhanced drug delivery into the human brain in order to develop new and improved methods for NP preparation and usage in CED to treat brain tumors and neurodegenerative diseases. • Apply the use of superparamagnetic nanoparticles to the CED research currently being preformed in LPPD. • Develop feasible scale-up methods for nanoparticle production.

32. References

33. Questions?